Immunological aspects of demyelinating diseases.

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lmmunol. 1992. 10: 153-J37

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IMMUNOLOGICAL ASPECTS OF DEMYELINATING DISEASES! Annu. Rev. Immunol. 1992.10:153-187. Downloaded from www.annualreviews.org by Stanford University - Main Campus - Lane Medical Library on 09/27/12. For personal use only.

Roland Martin, Henry F. McFarland, Dale E. McFarlin Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892 KEY WORDS:

demyelinating disease, multiple sclerosis. myelin basic protein

Abstract

Primary demyelination in the central nervous system results from damage to the myelin sheath or oligodendroglia and can be produced by a variety of mechanisms, including metabolic disturbances, toxicities, infection, and autoimmunity. The major human demyelinating disease affecting the cen tral nervous system is multiple sclerosis (MS). Although the etiology of MS is not known, existing data indicate that both genetic and environ mental factors contribute to pathogenesis. Experimental allergic enceph alomyelitis (EAE) is induced by immunization of genetically susceptible animals with myelin proteins. This is mediated by autoimmune T cells. Characterization of MHC restriction, fine specificity of antigen recog nition, and T cell receptor (TCR) usage by encephalitogenic T cells has resulted in highly specific immunotherapies. Both HLA and TCR genes have been linked to susceptibility for MS which is widely believed to be mediated by T cells that recognize an as yet unidentified autoantigen. Because of the advances in the understanding and treatment of EAE, recent research in MS has been focused on the characterization of cellular immune responses against myelin components. The results of these studies are reviewed and the potential implications of these findings for the patho genesis and future therapy of MS are examined. INTRODUCTION

The major human demyelinating disease is multiple sclerosis (MS). Although the pathogenesis and etiology of MS have not been established, I The

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Annu. Rev. Immunol. 1992.10:153-187. Downloaded from www.annualreviews.org by Stanford University - Main Campus - Lane Medical Library on 09/27/12. For personal use only.

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MARTIN, McFARLAND & McFARLIN

it is widely believed that immunological mechanisms are involved. In addition to MS, there are other disorders that affect myelin, and under standing of the pathogenetic processes involved in these conditions may have implications for MS. This chapter reviews the causes of human demyelinating disease in general and focuses on the recent studies of MS. Historically the concept of a demyelinating disease evolved from patho logical studies showing loss of myelin, with relative sparing of axons and neurons ( 1 ). In addition, the distribution of lesions in such diseases tended to be perivenous. The application of X-ray diffraction and electron microscopy to the investigation of the normal nervous system led to the identification of the lamellar structure characteristic of myelin (2). It was soon recognized that the myelin membrane is composed of lipids and protein forming a cover around axons and that myelin is produced in the peripheral nervous system (PNS) by Schwann cells (3) and in the central nervous system (eNS) by oligodendrocytes (4). A number of morpho logical, chemical, and antigenic differences between these two types of cells and between PNS and eNS myelin have been identified. However, the function of the myelin in both the eNS and the PNS is to increase the conduction of nerve impulses (5). In nonmyelinated axons, the action potential is transmitted by depolarization along the axonal membrane. This proceeds considerably more rapidly in myelinated fibers because the nerve impulse is propagated from one node of Ranvier to the next through the process of saltatory conduction. Clinical manifestations of demyelination vary and reflect dysfunction of the affected pathways. For example, if motor pathways are involved, then disturbances of movement occur clinically; similarly, demyelination of sensory pathways leads to abnormalities of sensation, etc. Thus, demy elination can result in a wide range of neurological abnormalities. These are associated with prolongated conduction of nerve impulses which can be demonstrated by physiological studies. Primary demyelination can result from damage to the myelin sheath or myelin forming cells. Because of interactions between the axons and myelin producing cells, destruction of the former can also lead to myelin loss. This is known as secondary demyelination or Wallerian degeneration (6). This chapter focuses on disorders that produce primary demyelination of the eNS. CLASSIFICATION OF DEMYELINATING DISORDERS

A variety of approaches have been used to categorize the demyelinating disorders (1, 6, 7). These include a simple listing of the different conditions (7) and classifications based on pathological (1) or etiologic aspects (6).


IMMUNOLOGY OF DEMYELINATING DISEASES

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Although all approaches are imperfect, the authors find it useful to con sider these disorders in terms of two variables: First, we must ask whether the PNS or eNS is primarily affected. Many disorders, characteristically involve either the PNS or the eNS, but some processes affect both. This distinction can be made by clinical and laboratory findings. Second, the disorder can be classified according to its possible etiology. Although the cause of many demyelinating disorders is not understood, sufficient advances have occurred to attempt this approach. A number of mech anisms, including metabolic, toxic, infectious, and immunological pro cesses can produce primary demyelination.

Metabolic Disorders Some of the genetically determined disorders affect the eNS white matter and produce varying amounts of demyelination. These include the sphingolipidosis in which a specific lipid accumulates because of a genetic defect resulting in a deficiency of an enzyme involved in catabolism (6). Many of these conditions have murine counterparts that are particularly valuable in the investigation of molecular mechanisms related to myelino genesis. The metabolic disorders of myelin have been reviewed elsewhere (8), but one disorder, adrenoleukodystrophy (ADL) (9), is relevant to considerations in this chapter. ADL is an X-linked disorder that presents in infantile to juvenile boys. Pathologically, there are large demyelinating lesions and perivenular infiltration of lymphocytes. In addition, increased production of immunoglobulin appears in the eNS ( 1 0). These findings suggest that an immunologic component is involved in the pathogenesis even though a genetically determined defect in the oxidation of long-chain fatty acid has been shown. A clinical variant known as adrenomyelo neuropathy occurs in older individuals (9). It is predominantly manifested by spinal cord dysfunction and has a slower clinical course than the juvenile form. A significant proportion of female carriers show various degrees of clinical abnormalities, and some of these have been misdiagnosed as MS.

Toxic Etiologies Demyelination may also result from toxic substances. Usually both pri mary and secondary demyelination are present, indicating that toxic sub stances affect axons or neurons, in addition to myelin or oligodendrocytes. However, some experimentally produced toxic disorders are mostly mani fested by primary demyelination. Two examples are the experimental disorders induced by cuprizone ( 11), a chelator used as a reagent for copper analysis, and triethyltin ( 1 2). These experimental diseases provide evidence that toxic substances can lead to demyelination. In some human disorders such as Marchiafava-Bignami disease and central pontine myelinolysis

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manifested by focal demyelination in specific areas of the eNS, a toxic contribution to the etiology has been proposed (7).


Infectious Diseases HUMAN DISEASES The best example of an infectious etiology for demy elination is progressive multifocal leukoencephalopathy (PML). This dis order is due to infection of oligodendroglia by a papovavirus ( 1 3) and usually occurs in immunocompromised individuals. PML was originally observed in patients who either had malignancies of the reticuloendothelial system or were immunosuppressed for therapeutic reasons, but in recent years PML has occurred with increasing frequency in patients with the acquired immune deficiency syndrome (14). Considerable attention has been given to a neurological disorder associ ated with human T lymphotropic virus I (HTLV-I). This condition, known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/ TSP), was originally observed in geographic regions where HTLV-I infec tion is endemic ( 1 5, 1 6). Only 1-5% of seropositive individuals de velop the neurologic syndrome. Although it mimics the progressive form of MS, there are a number of differences ( 1 7). The blood and cerebrospinal fluid (CSF) contain high titers of HTLV-I-specific antibodies, and proviral genome can be detected in cells of affected individuals by polymerase chain reaction (PCR). The major pathological findings are chronic meningitis and perivascular inflammatory infiltrates in the CNS. Demyelination is present but usually associated with loss of axons. High precursor fre quencies of CD8 + , HLA-class I-restricted, cytotoxic T lymphocytes (CTL) specific for regulatory or structural viral proteins are present in the blood and CSF. These correlate with the CNS disease, suggesting that immunopathological mechanisms are involved in the pathogenesis of HAM/TSP (18). Postinfectious encephalomyelitis (PIE) occurs following infection with a number of viruses, including measles, vaccinia, and varicella ( 1 , 1 3, 1 9). Attempts to isolate a virus from the CNS have been unsuccessful, and it is believed that the initial virus infection "triggers" an immune-mediated process that produces demyelination. This concept is supported by the demonstration of T cells that react to myelin basic protein (MBP) in individuals with PIE, following measles virus infection ( 1 9). ANIMAL MODELS A number of spontaneous and experimental models of virus-induced demyelination have been studied. Visna, a lentivirus-induced leukoencephalomyelitis of sheep, is perhaps the best spontaneous model for human disease (20, 21). This slowly progressive paralytic disease affects brain and spinal cord. The pathology consists of chronic inflammation



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and demyelination. Infectious virus cannot be isolated from the eNS; viral genome is, however, demonstrable in 1/104 cells (13, 21). Infected mononuclear cells are believed to carry virus into the eNS. Temporary reduction in eNS inflammatory infiltrates during immunosuppressive treatment suggests that the demyelinating process is partly immune mediated. Another well-studied model involves an animal disease caused by Theiler's murine encephalomyelitis virus (TMEV), a picornavirus, similar to human poliovirus ( 1 3 , 22, 23). Intracerebral inoculation usually leads to an acute paralytic disease (13, 22, 23) due to lysis of motor neurons. Some animals survive and develop a progressive inflammatory and demy elinating myelopathy ( 1 3, 22, 23). Susceptibility for the disease correlates with the development of a strong DTH response mediated by TMEV specific, class II-restricted T cells (23), and the late demyelination can be prevented by cyclophosphamide; these facts indicate that the myelin destruction is immune-mediated (24). Lymphokine-activated macrophages have been postulated to induce bystander demyelination. However, the demonstration that TMEV produces demyelination in nude mice demon strates that humoral immune mechanisms are also involved ( 1 3). The recent report of a monoclonal antibody that reacts with both TMEV and myelin (25) supports the view that B and T cells contribute to the demyelinating process. The neurotropic mouse hepatitis virus (JHM) produces a monophasic demyelination by direct infection of oligodendrocytes, but rat-adapted JHM virus induces a relapsing illness in Lewis (Le) rats ( 1 3 , 26). This can be passively transferred to naive animals by MBP-sensitized T lymphocytes derived from acutely infected animals, and the infection results in a dis order similar to experimental allergic encephalomyelitis (EAE) (26). Because MBP-specific T cells are generated during the initial phase of the disease, it is believed to represent an animal model for PIE. Experimental measles virus infection also leads to an expansion of MBP-specific T lymphocytes (27). This could be due to the release of myelin or antigenic cross-reactivity between viral and myelin proteins (26, 27), but evidence of the latter has not been documented.

Autoimmune Etiology Postvaccinal encephalomyelitis (PVE) was observed within a few years after rabies virus vaccination was introduced by Pasteur (28). In 1933, Rivers reproduced PVE in monkeys by the administration of the Semple vaccine (29). This vaccine was prepared by growing the virus in rabbit spinal cord; "control animals" that were immunized with normal spinal cord also developed PVE. Both the experimental disease and PVE are

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characterized pathologically by perivenular inflammatory reaction and demyelination. The experimental disease subsequently became known as EAE and has been extensively studied. LESSONS FROM EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS


Variables in Disease Production After Rivers' original description, a number of different EAE models were reported (30-32). These vary in manifestations, pathology, and patho genesis. Important variables in these experimental diseases are the species and strain of experimental animals, the material used for immunization, and the type of adjuvant employed. EAE has been produced in a number of species. In outbred animals, susceptibility and manifestations of the disease differ considerably (3032). In general, there is less variation among inbred species, and susceptible as well as resistant inbred strains of guinea pigs, rats, and mice have been identified (32). In Rivers' experiments, the disease was produced by multiple injections of rabbit spinal cord. After the introduction of adjuvants, it became pos sible to generate EAE after a single immunization (3 1). This facilitated the identification of encephalitogenic components that reside in the white matter and are a component of myelin. Subsequently, MBP was identified as the major encephalitogen (32). This protein has been extensively studied and is conserved among most species (32). Different molecular forms occur and are generated by differential splicing of seven exons from a single gene (33). In addition, there are substitutions and deletions that can affect encephalitogenic activity (30, 32). The encephalitogenic epitopes vary among certain species and even in different strains of a given species. The general characteristics of myelin proteins as well as the major enceph alitogens are summarized in Table 1 . A second myelin component, the proteolipid protein (PLP), is enceph alitogenic in some species (Table 1 ) (35, 36). This protein is highly hydrophobic and difficult to work with (35). Some of the technical diffi culties have been overcome by using synthetic peptides (36). Although less extensively studied than MPB, it is already apparent that PLP contains more than one encephalitogenic region. There are many other proteins in the oligodendrocyte/myelin membrane that could function as target antigens. These include the myelin-associated glycoprotein (MAG) (37) and myelin oligodendroglia protein (MOG) (38). These proteins, however, have been studied less extensively (see Table 1). Immune reactivity against lipids also occurs and may contribute to the autoimmune process in the


Table 1

Myelin antigens and encephalitogenic epitopes in EAE'

Protein (Ref. %)

Size (kDa)

Myelin basic protein (MBP; 30%)

18.5

Encephalitogenic epitope(s) 1-9Ac, 35-47 89-100, 92-10 1 , 96-109, 1 7-27 69-86, 72-84 87-99 (human) 1 53- 1 65

Proteolipid-protein (PLP; 50%)

30

Myelin associated glycoprotein (MAG; 1 %)

1 00

Myelin oligodendro glia glycoprotein (MOG)

54

•

Whole protein 1 39- 1 5 1 , 1 4 1-1 51 103-1 1 6

Animal strain

Restriction element(s)

BIO.PL mice PLj J mice SJL/J mice

lA", IEu lAu lA'

Lewis rats

lA (RT-l)' IE

Localization Cytoplasm of oligodendroglial cells

Rhesus monkey SJL/J mice

lA'

SWR

lAq

Not known

Not known

The table refers to the major encephalitogenic epitopes and the most widely used animal strains.

Remarks Charge isomers are generated by posttransla tional modifications; size isomers of 14.0, 1 7 .2, 1 8 . 5 and 2 1 .5 kDa

Reference (30,59-6 1 ) (30,34) (30,62) ( 1 36) ( 1 37)

Highly hydrophobic; interacts with lipids

(35, 36)

Myelin sheath membrane (periaxonal space)

Member oflg supergene family; shared epitope with NK-cells

(37)

Myelin membrane at the surface of oligodendrocytes

Anti-MOG antibodies increase the severity of MBP-induced EAE

(38)

Myelin membrane

SE

�

Z 0 t"" 0 Cl ...:: 0

'" ..,

Z

Cl

I:j t;J m > '" m '" -

VI \J:)


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CNS when whole tissue or myelin is used for immunization; however, except for isolated reports, EAE has not been produced by immunization with lipids alone (39). In most models of EAE, disease has been produced by immunization with various encephalitogens in Freund's complete adjuvant. The addition of Bordetella pertussis gives rise to a more acute disease in Le rats (40) and is an essential for the production of disease in one murine model (41).

Variables in Disease Manifestation EAE has been investigated by scientists in a number of disciplines. Neuro scientists have been interested in clinical and pathological aspects of the disease that mimic human disorders, particularly MS. EAE has provided immunologists with a T cell-mediated disorder for the investigation of T cell recognition and regulation in a pathological process. Typically, EAE is monophasic, but chronic relapsing models have been developed (30) and provide the basis for the study of in vivo immune regulation as well as experimental therapeutic manipulation. The pathology of EAE consists of perivascular inflammatory cells; how ever, the cellular composition and extent of demyelination vary in the different models. For example, EAE in Le rats produced by immunization with MBP is predominantly monophasic, and the major pathologic feature is perivascular inflammation with minimal demyelination (42). In com parison, in guinea pigs there is more demyelination and, when the im munogen was supplemented with glycolipids including galactocerebroside, demyelination was even more prominent (43). These observations have led to the concept that the initial process is mediated by a T-cell response to MBP and the demyelination by antibody to a lipid (43). However, this probably varies among species because some demyelination in addition to perivascular inflammation occurs in mice after immunization with MBP alone.

EAE as a T Cell-Mediated Disease The demonstrations thatEAE could be adoptively transferred by immune cells in rats (44) and guinea pigs (45), but not by serum, provided direct evidence that the disease is cell-mediated. Methods that augment the transfer of the disease in rats and mice were developed (46-47), and subsequently, encephalitogenic T cell lines (TCL) and clones were pro duced (48), demonstrating that EAE was transferred and presum ably mediated by CD4 + T cells (49). Knowledge of the biology and func tion of this T-cell subset has led to different strategies for modifying EAE.


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Prevention of EAE by Blocking Antigen Presentation Treatment with antibody to class-II MHC molecules prevents EAE, pre sumably by altering presentation of encephalitogenic epitopes to CD4 + cells (50). Recently, a monoclonal antibody specific for antigen-presenting molecules pulsed with encephalitogenic epitope has been produced that blocks the disease in SJL mice (5 1 ). Another approach has been to use nonencephalitogenic peptides that closely correspond to an enceph alitogenic epitope (52, 53). In PL mice, a major encephalitogenic epitope resides in the nine N-terminal amino acid residues of MBP (Table 1 ). Acetylation of the N-terminal alanine is essential, and treatment with a . nonacetylated peptide inhibited the induction of the disease (52, 53). In a subsequent study, a series of synthetic peptides with alanine substitutions at each of the nine residues were tested for the capacity to react with (H2U) class-II molecules and to stimulate T-cell clones (53). Peptides were identified that bound to I-All but were not recognized by immune T cells. Treatment of PL mice with a synthetic peptide with high affinity for class II molecules blocked the adoptive transfer of EAE by peptide-specific clones, presumably because of competition with the encephalitogenic pep tide for class-II molecules. EAE has also been treated with unrelated peptide with high affinity for lAS (54) and with a random copolymer of four amino acids designated Copl (55). The latter may either alter antigen presentation or induce suppressor cells.

Treatment of EAE by Altering T-Cell Function Historically, EAE can be prevented and blocked by a variety of immuno suppressants including antilymphocyte serum. After the disease was shown to be mediated by CD4 + T cells (49), antibodies against this subset were demonstrated to reverse EAE in rats (56) and in mice (57). These approaches have the potential to produce general immunosuppression, and more specific strategies have been developed. One way has been to induce a state of unresponsiveness to the encephalitogenic epitope (58). In these studies, encephalitogenic antigens were coupled to carrier cells that were injected into animals before or after induction of EAE. This modified both induction and effector phases of the disease (58). Another specific treatment of considerable interest is to administer antibody to the T cell receptor (TCR) expressed by encephalitogenic T cells. In some susceptible strains of mice including the PL and B 1 O.PL, the TCR chain Vf38 is expressed on 1 6-25% of peripheral T cells (59, 6 1). When lymph node cells from PL mice immunized with the encephalitogenic peptide 1-11 NAc were sorted by FACS into Vf38 + and Vf38- cells and assessed for the capacity to proliferate in response to the encephalitogenic


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peptide, the responding cells were V[38 + (59, 60). Subsequent molecular studies showed that T-cell clones specific for peptides 1-9 NAc pre dominantly used V[3S.2 and Va4.3 (59, 60). The usage of D[3 and J[3 was also limited, but to a lesser degree. Four clones used J[32.7, while J[32.3 and 1[32.5 were each used by two clones (60). AIl S clones used Va4.3 (60). The TCR used for the recognition of peptide 1-9 NAc was also examined in T-cell hybridomas derived from B l O.PL mice (61). Th�s strain also develops EAE in response to peptide 1-9 NAc, and 26/33 of the hybri domas used V[3S.2 and 1[32.6. Furthermore, all 33 hybridomas used either Va2.3 (61%) or Va4.2 (39%) (61). Following the analysis of TCR usage for recognition of encephalitogenic peptide, treatment with monoclonal. antibodies specific for V [3S.2 was attempted. EAE was produced in PL mice by adoptive transfer of a VpS.2+ encephalitogenic T-cell clone. Treatment with monoclonal antibody 23.1 that depletes V[3S.2+ T cells resulted in a striking reversal of neurological dysfunction (59, 60). Admin istration of antibody 23.1 also prevented the induction of EAE after immunization with MBP peptide 1-11 NAc in CFA (60). Similar results were obtained in B l O.PL mice, and it is noteworthy that encephalitogenic cells directed at other epitopes did not appear in these animals (61).

T-Cell Vaccination Initial studies of vaccination with disease-producing TCL and clones were conducted in Le rats (62). Long-term encephalitogenic CD4+ TCL were inactivated by treatment with pressure or fixatives and used in CFA for vaccination of normal rats (62). These animals became resistant to EAE produced by either direct immunization or adoptive transfer. Subsequently, Le rat T cells were shown to use VpS.2 for recognition of the encephalitogenic epitope (63). This led to vaccination with a synthetic peptide corresponding to the amino acid (AA) sequence of the comple mentarity determining region 2 (CDR 2) of Vf38 (64). The vaccinated Le rats did not develop EAE after challenge by MBP, but produced antibody against Vp8.2 + and CD8 + T-cells that recognized the Vp8.2 peptide in association with class-I MHC (65). Based on studies of the interaction of TCR with antigenic peptide, it is believed that the CDR2 region of the V chain primarily interacts with the MHC molecule (66). Since the CDR3 Gunctional) region is specific for each TCR molecule and probably forms contact with the antigenic peptide or a combination of the latter with MHC, peptides homologous to this region of the TCR chain were used for vaccination and protected PL mice from the disease (67). A goal of T-cell vaccination studies is to provide the background for experimental therapeutic responses in MS discussed below. This disorder presents clinically long after the putative immunopathologic processes


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have been initiated. Recently, vaccination of Le rats with VfJ8.2 peptide after onset of EAE has been studied (65). The findings indicate that the duration and severity of the disease can be reduced by the vaccination protocol. In addition to preventing the autoimmune disease, these obser vations provide direct evidence of an immune network.


Prevention of EAE by Oral Tolerance The report that contact sensitivity can be prevented by the oral adminis tration of simple chemicals led to attempts to modify EAE. In Le rats, the feeding of MBP in large amounts induces resistance to EAE (68, 69). This protection has been reported to be transferred by CD8 + T cells (69). It has recently been shown that the CD8 + T cells released a cytokine, possibly transforming growth factor f3 (TGFf3) that downregulates EAE CR. L. Weiner, personal communication). At present, it is not clear whether the effect of oral tolerance is only mediated by anergization of autoreactive T-cells (70) or by the induction of suppressor mechanisms such as TGFf3.

Inhibition of EAE by TGF{3 In order to employ the highly specific forms of immunotherapies such as T-cell vaccination and interference with antigen presentation described above, knowledge of encephalitogenic epitopes, TCR usage for recog nition, and MRC molecules involved in presentation is essential. However, an alternative approach is to use strategies that downregulate immune reactivity. In this regard, it is of considerable interest that the ad ministration of small amounts of TGFf3 inhibits EAE in both rats and mice (71). In addition, when it was given to mice with chronic relapsing disease, the severity of the clinical course was drastically reduced (7 1). MULTIPLE SCLEROSIS

MS most commonly affects young adults. The clinical course is quite variable, but the most common form is manifested by relapsing neurologic deficits. Recent findings with magnetic resonance imaging indicate that considerable subclinical disease occurs and that, early in lesion develop ment, there is a breakdown in the blood brain barrier (BBB) (72). Although the pathogenic mechanisms responsible are not understood, it is widely believed that MS has an immunopathologic basis and that both genetic and environmental factors contribute.

Evidence of an Immune-Mediated Process The evidence supporting an immune-mediated mechanism in MS is cir cumstantial and includes the following: .

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1. The pathology of the lesion. 2. Similarities with PVE and EAE. 3. Increased disease activity following administration of interferon y (lFN-y). 4. Immunological abnormalities. 5. Immunogenetic background. The characteristic lesion in MS is the plaque; this refers to the macroscopic appearance of a demyelinating lesion. On gross examination, plaques are sharply demarcated from the surrounding tissue and vary in color depending on the age of the lesion (73). Fresh inflam matory foci are reddish, whereas older ones appear grayish. Plaques occur throughout the white matter of the CNS, but predilection sites are well known. Microscopic examination shows areas of circumscribed demyelination that generally contain intact axons. Viable oligodendrocytes are present in early stages of plaque development but later decrease in number (42, 73). Inflammatory infiltrates are located perivenularly, and the cellular composition of infiltrates varies with the age of the lesions. Acute lesions consist of macrophages, lymphocytes, plasma cells, and bare non myelinated axons (42, 72). The phenotype of the T-cell infiltrate in lesions has varied among different studies. Overrepresentation of CDS + in com parison to CD4 + cells has been reported, but a preponderance of CD4 + cells at the edge of the plaque and in the surrounding parenchyma was observed in other studies (74-76). These variations may be related to technical or sampling differences. T lymphocytes express activation mol ecules on their surfaces such as IL-2 receptors and class-II HLA molecules (77). Another sign of the ongoing immunological process is the expression of HLA-class II molecules not only on macrophages and T cells, but also on resident CNS cells including brain capillary endothelial cells, microglia, and astroglial cells (7S). This is probably mediated by IFN-y secreted by activated T cells. Ultrastructural studies indicate that macrophages are primarily involved in the initial steps of myelin destruction. Myelin lamellae are stripped from the axon by an unusual phagocytic mechanism that starts with attachment of superficial lamellae to vesicles at the macrophage surface that are called coated pits (42, 73). Immunoglobulin deposition between macrophages and myelin lamellae has been described and may contribute to the process (42, 73). Complement and proteolytic enzymes generated by macrophages are probably involved in the terminal steps of myelin destruction (73, 79). In summary, the nature of early MS lesions indicates that demyelination occurs in the presence of an active immune response, although the antigen

NATURE OF THE LESION



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responsible for this is not yet known. In chronic plaques, inflammatory infiltrates are less pronounced, and there is reactive gliosis and abortive attempts at remyelination. Recently, the presence ofT cells expressing ylb heterodimers of the TCR and the presence of heat shock proteins in close proximity have been described (80). These findings suggest that the immunological mechanisms in more chronic lesions are different from those in acute lesions. Collectively, the pathological observations are con sistent with the concept that different immunological processes occur as the disease enters a chronic phase. Histopathologic similarities between PVE and MS support the hypothesis that the latter is immune-mediated. PVE is characterized pathologically by perivascular inflammation and demyelination. The demyelinating lesions tend to be periventricular like those in MS ( 1 , 7). Many of the lesions are large enough to be visualized

SIMILARITIES BETWEEN PVE AND EAE

macroscopically. These are probably formed by the confluence of small

lesions. There are some differences between PVE and MS. In particular, the lesions in PVE tend to have the same age, whereas lesions in MS characteristically vary in age ( 1 3, 42). Although a definitive treat ment for MS has not been established, over the past decade there has been an increase in the experimental use of therapeutic agents. Safety factors are incorporated into therapeutic trials, and an open pilot study of the use of IFN-y was terminated because 7 of 1 8 patients treated with this agent experienced a clinical exacerbation within one month after initiation of treatment (8 1). This was accompanied by a high rate of spontaneous proliferation of peripheral blood lymphocytes (PBL) and an increase in the specific response to MBP.

EXACERBATION AFTER TREATMENT WITH INF-y

Recent years have seen intensive inves tigation of various immunological parameters in MS. Each new advance in immunology has subsequently been applied to the study of MS. Despite efforts by many investigators, however, an immunological abnormality responsible for the pathogenesis of MS has not been identified. Reder & Amason (82) recently reviewed in detail the various immunological find ings. This extensive material is not repeated here, but the authors' con clusions deserve restatement: "A sense emerges that something is awry with immune function in MS" and "the various abnormalities [that] have been discussed, while tantalizing, offer no firm clues as to the cause of MS" (82). Several possible explanations for the difficulties in formulating a hypoth esis for the pathogenesis ofMS can be offered: First, although considerable IMMUNOLOGICAL ABNORMALITIES


166


evidence suggests that MS is an immune-mediated disease, the antigen or antigens against which the immune response is directed are not known. Secondly, considerable variation appears in the disease and, on occasion, investigations may not have focused on the appropriate subgroup. Thirdly, as emphasized above, MS is limited to the CNS, and most studies have been conducted with blood. Whether abnormalities found with PBL reflect the disease processes in the brain is not clear. In spite of these difficulties, some findings that have been reported consistently over the years are discussed. Immunoglobulin synthesis in the eNS Increased amounts of immuno globulins in the CSF have been consistently demonstrated, since Kabat's original description in 1950 (83), and this finding is currently used as a diagnostic criterion by many clinicians. The elevated CSF IgG is due to increased synthesis of IgG, and in some patients, IgM and IgA are also elevated (84). Separation of IgG by electrophoresis or isoelectric focusing characteristically shows a limited number of distinct bands (85). These so called "oligoclonal bands" are observed in more than 90% of patients and are also used as a diagnostic aid for MS. A major unanswered question is: What is the mechanism responsible for the increased production of immunoglobulins in MS? Elevated immunoglobulins occur in the CSF of other disorders including syphilis, subacute sclerosing panencephalitis, Lyme disease, and HAM/TSP ( 1 7, 84, 86). However, in these conditions, the immunoglobulin production is antigen driven and directed at the causative infectious agent(s). Numerous studies have been conducted to determine the specificity of the increased CSF immunoglobulins in MS, but to date a single antigen or infectious agent with which these react has not been identified. There are at least two possible explanations: The first is that MS is caused by an unidentified infectious agent, discussed below, and that the increased immunoglobulins are directed against this. The second is that MS is related to a defect in immune regulation, and the increased production of immunoglobulins in CSF simply reflects this abnormality.

Evidence of impaired suppressor function in MS was originally suggested by the study of a mitogen-driven assay system that was used to generate a soluble suppressor activity (87). This approach indicated that the mitogen-induced suppressor activity was reduced during acute exacerbations of the disease. In subsequent studies, PBL derived from MS patients and stimulated with pokeweed mitogen produced larger quantities of immunoglobulins than those from controls (82, 87). This could be in part corrected with mitogen-induced suppressor factors from T cells and, consequently, it was postulated that the overImpaired immune regulation


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reactivity ofB cells was related to impaired suppression. Initially, the defect was thought to represent abnormal function of CD8 + cells; however, an alternative hypothesis has been that the defect is related to a reduction in a subset of CD4 + cells, designated suppressor-inducer cells (88). Another indication that CD4 + cells are abnormal is that most MS patients have reduced numbers of measles virus-specific CTL that are CD4 + (89). GENETIC FACTORS Support for a genetic influence in MS has come from the investigation of prevalence in different ethnic groups as well as from family studies (90-95). In Northern American and Northern European Caucasians, the disease is common, with a mean prevalence of about 60. (All prevalences are expressed x 10- 5.) However, it is virtually absent in certain ethnic groups like Hutterites, Yakuts, Inuit, or Bantu (90). Geographic differences in prevalence could reflect both environmental and genetic factors; thus, the prevalence in genetically homogenous groups that have migrated is of interest. Relative low prevalence of 2.0-4.0 is found not only in Japan, but also in Japanese living along the Pacific Coast of the United States or in Hawaii (90). In addition, Hungarian gypsies show a prevalence rate of 2.0 compared to 37 in the general Hungarian population (9 1). Although these differences in prevalences are generally thought to reflect the unique genetic background of the distinct groups, environmental factors including different life styles and nutrition could partly account for the findings. Additional support for theories about the influence of genetic factors comes from family studies. Familial cases of MS have long been known, and various studies have observed that between 1 2.9% and 19% of indi viduals with MS have affected family members (92, 93). First-degree rela tives carry the highest risk, and more distant family members are affected less frequently (92). These conclusions were based on clinical observations, and many "normal" family members show evidence of "subclinical MS" when laboratory investigations such as neurophysiological, magnetic res onance imaging, and CSF are included. The strongest evidence of a genetic influence comes from comparison of concordance in monozygotic (MZ) and dizygotic (DZ) twins. A higher concordance has been found consistently in MZ twins (94, 95); however, the degree of concordance has varied considerably, and many early studies were flawed by ascertainment bias. A recent population-based study designed to minimize ascertainment bias demonstrated concordance in 26% of MZ and 2.3% of DZ twins (95). MRI studies showed lesions consistent with MS in two fifths of the "clinically normal" monozygotic twins of individuals with MS. Since the mean age of the twins at the time was only around 30 years, many of the unaffected twins are still at risk to


1 68


develop the disease (95). Although the results from family and twin studies clearly indicate that genetic factors influence susceptibility to developing MS, concordance does not reach 1 00% in MZ twins. Thus, the disease is not simply genetically determined. Instead, the development ofthe disorder is believed to be the consequence of multiple genes (see below) plus an environmental factor. Association with MS was demonstrated for a num ber of genes, i.e. for alpha antitrypsin, GM allotypes, and the 5' flanking region of the MBP gene (96-98), but the focus of the search has been on the HLA and the TCR gene complex. HLA Genes First reports that HLA-A3 and -B7 are associated with MS in Northern American and Northern European MS populations date back to the early 1 970s (99). Analogous to other autoimmune diseases and EAE, stronger associations have, however, been observed with HLA-class II genes, namely HLA-DR21 ( 100). In ethnic groups with a lower prevalence of MS, associations with other HLA-DR genes have been reported: HLA DR4 is associated with the disease in Italians and Jordanian Arabs ( 1 02, 1 03), and HLA-DR6 in Japanese and Mexican MS populations ( 1 04, 105). The observation of significantly higher prevalences in DR2-positive Hungarians (9 1), compared to HLA-DR2 positive gypsies living in the same area, indicates that other predisposing or even protective factors must exist or that different DR2 fJ-chains are expressed in the two populations. Compared to HLA-DR2, a relatively lower and probably independent association with HLA-DPw4 has been described in two Scandinavian studies ( 1 06, 1 07). The recent investigations of HLA-antigens at the molecular level have identified restriction fragment polymorphisms (RFLP) associated with MS ( 1 08- 1 1 3). These studies indicate that HLA-DQB l (HLA-DQBI 0602) genes confer a higher risk than does HLA-DR2 alone ( 1 08, 1 09). Because the HLA-DQB l and HLA-DR2 genes are part of the same haplotype and in close linkage dysequilibrium (WI), it is difficult to assess whether the two genes confer susceptibility together or independently. A Norwegian study of 6 1 MS patients showed that 97% were positive for the HLA DR antigens (HLA-DR2 Dw2, HLA-DR4, or HLA-DR6), previously associated with the disease ( 1 09). Moreover, comparison of the DQfJ chains linked with these HLA-DR molecules indicated that most of the AA sequences in the membrane distal domains of DQfJ were shared. This led the authors to propose that the polymorphic region of the DQfJ chain that, together with DQa, contributes to the formation of the binding groove presents a putative autoantigen related to the disease. Another I The serological specificity (using new nomenciature-IOI), associated with MS is DRw 1 5, a recent split of DR2. The D specificity determined by lymphocyte typing is Dw2.



169

report detected an informative DQoc RFLP that confers risk independent from DQfJ ( 1 1 0). This was identified in pooled DNA collected from MS patients in Northern Ireland and Scotland, and digested with the enzyme Msp1 ( 1 1 0). Although linkage between MS and certain HLA antigens has been consistently demonstrated, the mechanism for this is unknown. Recently, exon II encoding the hypervariable regions of DQoc, DQf3, and DRf3 in 6 MS patients have been sequenced, and unique sequences that correlate with disease, analogous to insulin dependent diabetes, have not been found (Il l ) . Another possibility is that a separate gene on chro mosome 6, located in the neighborhood ofHLA-DR and -DQ genes, could be involved in susceptibility. Consideration has been given to the possibility that different clinical courses of the disease such as relapsing remitting or chronic progressive MS correlate with the immunogenetic background ( 1 1 2, 1 1 4). An association between HLA-A3, -B7, and -DR2 and the relapsing form was reported, while patients with progressive disease were HLA-A1, -B8, and -DR3 ( 1 14). This question was pursued further by analyzing the HLA-class II gene polymorphisms in 100 Scandinavian MS patients (1 1 2). The DR2Dw2 and DQw6 haplotype was strongly associated with both chronic progressive and relapsing remitting forms of the disease, but in a group of 74 relapsing remitting patients, a positive association with a DQfJ RFLP was found in the DRw 1 7 DQw2 haplotype. In 26 chronic-progressive MS patients, additional risk was conferred by a DQf3 pattern present in the D R4 DQw8, DR7 DQw9, and DRw8 DQw4 haplotypes, whereas a pattern observed with the serological specificity DQw7 was negatively associated. Both RFLPs associated with chronic progressive and relapsing-remitting MS were allelic to the HLA-DR2 DQw6 haplotype. In summary, different HLA-DR specificities are associated with MS in different ethnic groups. This may mask the association with DQBl genes that encode shared AA sequences ( 109). In Caucasians, higher risk is conferred by HLA-DQw6 than by HLA-DR2 Dw2 ( 1 08-1 10, 1 12, 1 13). Additional risk may be conferred by another HLA-gene, which is either a DQoc ( 1 10, 1 13) or a second DQf3 gene (1 1 2). Whether one of the candidate autoantigens is binding to this HLA antigen or whether they are involved in regulation of the autoimmune response is not yet clear. Since linkage data, sib pair analysis, and twin studies indicate that multiple genes influence susceptibility to MS, inves tigation of the TCR gene complex was of particular interest. These inves tigations have addressed the question whether there are differences in MS patients and the general population with respect to the TCR germline repertoire. Analysis of the TCR fJ-chain germline repertoire was performed T-Cell Receptor Genes

170

MARTIN,

McFARLAND & McFARLIN

by southern blot using probes for the human V{3 gene families V/1 1 14 and the constant (C{3) region in 40 patients with chronic progressive MS and 1 00 controls ( 1 1 5) . Allelic forms ofV{38, Vf3 1 1 , and Cf3 allowed the assign ment of haplotypes, and one termed 23/20/9 according to the fragment sizes was overrepresented in the patient group, whereas the haplotype 2/25/ l O was underrepresented ( 1 1 5). Stratification of the patients to DR type and comparison of the DR2 + patients and controls disclosed that the 23/20/9 haplotype confers a relative risk of 3.22, indicating that one or more genes in the TCR f3-chain complex on chromosome 7 might contribute to disease susceptibility in addition to HLA-genes. These results were not confirmed by a Scandinavian study that used the same RFLP, indicating that the MS populations in the two studies were probably genetically different ( 1 1 6). The inheritance of TCR f3-chain genes was followed in 40 sibling pairs, concordant for relapsing remitting MS, by using TCR f3 chain RFLP ( 1 1 7). Individual haplotypes were assigned, and the number of haplotypes identical by descent in sib pairs was determined. In affected sib pairs the number of shared haplotypes was significantly higher than expected. Haplotype sharing was random, however, when MS patients were com pared with unaffected siblings. One TCR {3-chain RFLP generated by digestion with HindUI was overrepresented on chromosomes of MS pa tients, compared to parental chromosomes not transmitted to unaffected offspring ( 1 17). Similar techniques were employed to study TCR et-chain alleles in MS patients from California and Australia ( 1 1 8). Digestion of DNA with Pss1 and hybridization with cDNA probes for TCR Vet 1 2 . 1 and Cet detected polymorphic bands of 6.3 (Vet) and 2.0 Kb (Cet). The relative frequencies of the two RFLP in Californian controls were 0.3 and 0.44. Comparison of 28 Californian and 47 Australian MS patients with controls detected a strong association between the disease and the 2.0 Kb RFLP in both populations (relative risks: 1 6.53 and 16.4). In addition, a strong associ ation between the disease and the 6.3 Kb RFLP (relative risk 10.95) was found in the Californian but not the Australian MS patients (relative risk 2.0). The studies of the TCR germline repertoire suggest that genes within the TCR (X- and p-chain complex may confer risks that are independent from HLA genes. Before firm conclusions can be reached, confirmation in larger patient samples and in different ethnic backgrounds will be required. In addition, it is important to determine whether MS segregates with TCR germline genes in families with multiple affected members. Investigation of TCR usage in lesions and responses to autoantigens are described below.


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171

Recent Investigation of Autoantigen-Specijic T-Cell Responses Although the concept that disturbances in the regulation of autoantigen-specific T cells produce demyelination is widely held, a problem with this hypothe�s is that the responsible autoantigen is unknown. It is also possible that more than one antigen is involved. There is hope that the approaches used for the study of T-cell recognition and regulation in EAE will provide new answers. Consequently, a considerable amount of research in this area is currently in progress. To date, this work has primarily focused on MBP, but investigation of T-cell reactivity to other myelin components including PLP, MAG, and MOG, has shown increased numbers of cells responding to these myelin proteins ( 1 1 9121). Some of the characteristics of the most prominent candidate brain autoantigens are summarized in Table 1. Since the cellular immune response against MBP has been studied in greatest detail, the discussion here is focused on reactivity to this protein. Human MBP occurs in four isoforms of different molecular weights gen erated by alternative splicing of the mRNA (see Table 1). In addition, isomers that differ in charge are produced by posttranslational modi fications (122). Studies to date have used the most abundant and basic form of human MBP ( 1 8.5 kd; 1 70 AA) (32). Soon after techniques were developed to examine T-cell function and specificity, it was demonstrated that MBP-specific TCL can be generated from the PBL ofMS patients and healthy individuals ( 1 23). These observations were followed by extensive search for differences between MS patients and controls in immune reac tivity against MBP. The following questions have been of particular inter est: 1. Are the frequencies of MBP-specific T cells in MS patients similar to those of controls? 2. Do MBP-specific T cells from both populations differ in terms of fine specificity? 3. Which HLA determinants serve as restriction elements for MBP-specific T cells? 4. Is there a unique and restricted TCR us�ge in antigen-specific TCL or T cells in the brain in MS patients? Although none of these questions has been unequivocally answered, new information has emerged in each area.


ANTIGEN-SPECIFIC T CELLS

The studies reported so far have described significant increases in the precursor frequencies in the blood and CSF of MS patients (11 9, 1 24, 1 25). Moreover, in vivo-activated MBP-specific T cells could be expanded only from PBL of MS patients ( 126). When IFN-'l' release by MBP-stimulated cells was chosen as a readout, precursor frequencies of about 2.7-5.2 x 1 0-5 (PBL) and 1 85 x 1 0- 5 (CSF) were found (1 1 9). Lower precursor frequencies between 0.68 x 1 0-5-0.5 x 1 07 ( 125) of MBP-specific T cells were detected by FREQUENCY OF MBP-SPECIFIC T CELLS

172



lymphoproliferation. However, both approaches indicate that the number of MBP-specific T cells in the blood and CSF of MS patients is higher than in those of healthy individuals or controls. Although this is consistent with the concept that MBP is a target antigen, the possibility cannot be excluded that higher numbers of MBP specific cells in MS patients result from expansion of preexisting MBP�specific T cells following myelin release during the disease. FINE SPECIFICITY OF MBP-SPECIFIC T CELLS The fine specificity of a set of T-cell clones established from one MS patient was dissected by using a number of peptides from human and xenogenic sources ( 127). Ten different recognition patterns were observed, indicating that considerable hetero geneity existed in the fine specificity of human MBP-specific T cells ( 1 27). Subsequently, some of the M BP-specific CD4 + TCL were shown to exhibit cytotoxic activity, but fewer epitopes were recognized by cytotoxic TCL ( 128). Because studies of EAE demonstrated that encephalitogenic TCL can be derived from bulk cultures of lymphoid cells by repeated in vitro stimulations ( 129), a similar strategy was employed in MS patients and controls. TCL were established, and the fine specificity was assessed by both proliferation and cytotoxicity (130, 131). In one study, a more hetero geneous pattern of fine specificities was observed in the TCL from MS patients than from the controls ( 130). In a second investigation, MBP specific cytotoxic TCL were established from comparable percentages of MS patients and controls ( 13 1). Fine specificities were determined by using a panel of 30 enzymatically generated or synthetic peptides. When reactivities against large cathepsin-D derived peptides spanning AA 1-44, 45-90 and 91-172 2 were tested, significant differences between MS patients and controls were not detected ( 13 1). Two peptides located in the middle of the molecule (87-106) and at the C-terminus ( 154-172) which had been used previously ( 1 28) were recognized by a large fraction of TCL from both groups ( 1 3 1 ). The percentage of TCL responding to peptide 1-44 suggested the existence of a third major epitope in the N-terminal region (13 1). All of the MBP-specific TCL were CD4 + and secreted substantial amounts of IFN-y (13 1) and tumor necrosis factor ex (TNFex; R. Martin, R. Voskuhl, N. H. Ruddle, H. F. McFarland, unpublished observation). Thus, MBP-specific, cytotoxic human TCL are functionally similar to encephalitogenic T cells mediating EAE ( 132). In parallel, other investigators assessed the fine specificity of TCL estab2 The authors prefer to use the numbering of the porcine MBP sequence, since this is the longest mammalian MBP ( 1 72 AA) (30). However, different numbering has been used by other investigators and was not changed in this text.



1 73

lished directly from the peripheral blood ( 1 25, 1 33). The results were similar to those obtained with long-term TCL. Peptides 84-102 and 1 431 68 were recognized by a considerable number of TCL (125). In D R2 + MS patients, peptide 84-102 tended to be immunodominant, whereas 1 431 68 was recognized in comparable frequencies by TCL from both MS patients and controls in HLA-DRwl l + individuals. In another study, responses of TCL that were established using the cathepsin-D derived peptides ( 1 33) were examined. No significant differences in fine specificity or precursor frequency between patients and controls were found ( 1 33). Collectively, the data from various laboratories indicate that an immunodominant epitope exists in the middle of the molecule. The high number of TCL responding to peptides in that region (87-106, 1 3 1 ; 84102, 1 25; 80-99, 1 34) support this notion, and it is of interest that peptide 87-106 is recognized in the context of four HLA-DR molecules previously associated with MS in different ethnic groups (HLA-DRw1 5 Dw2; DR4 Dw4; DR4 Dw1 4; DRw 1 3 Dw1 9) ( 1 3 5). The minimal sequence of this epitope is located between AA 89-99 ( 1 35). Peptides 80-99 ( 1 34) and 841 02 ( 1 25) which were recognized in the context of one of the polymorphic beta chains of HLA DRw I 5, HLA-DR2b ( 1 34), and HLA-DQBl ( 125) probably all contain similar core sequences. Encephalitogenic epitopes for Le rats ( 1 36) and SJL mice are contained in this portion of the molecule (30). Recent investigations with CTL clones specific for peptide 87-106 demonstrated a number of nested epitopes within this sequence (R. Martin, U. Utz, J. E. Coligan, J. R. Richert, M. Flerlage, E. Robinson, R. Stone, W. E. Biddison, D. E. McFarlin, H. F. McFarland, unpublished results), similar to the encephalitogenic region in SJL mice (30). In addition, the other two major specificites, one at the C-terminus and the other at the N-terminus overlap with encephalitogenic epitopes in various animals, i.e. AA 1 53-1 65 in the rhesus monkey ( 1 37) and AA 1�9Ac in PL/J and B l O.PL mice (30, 59-61). As mentioned earlier, reactivity to the major molecular weight isoform of MBP has been analyzed so far. It is not known whether T-cell responses are also directed against epitopes encoded by exon 2 of the MBP gene. Exon 2 is not expressed in the 1 8.5 Kd MBP. Quite recent investigations have shown cellular immune reactivity against citrulline-containing epitopes that are generated in charge isomers ( 1 38). These are not found in the more basic major isoform of MBP, and it is conceivable that a response to such epitopes is unique to MS. HLA restriction of TCL has been studied by antibody blocking of proliferative or cytotoxic activity ( 125, 1 30, 1 3 1 , 1 33) and the use of well-defined homozygous HLA RESTRICTION O F HUMAN MBP-SPECIFIC T CELLS


1 74


typing cells for antigen presentation ( 1 3 1). In addition, mouse and human fibroblasts transfected with various HLA-DR r:J. and f3 chains have been used in cytotoxicity and proliferative experiments ( 1 3 1 , 1 34, 1 35). The majority of long-term as well as short-term TCL were restricted by HLA DR molecules ( 125, 1 30, 1 3 1 , 1 34, 1 35). HLA-DQ or -DP restriction has been rarely observed in proliferative TCL ( 1 25, 1 30, 1 33). HLA-DR2 showed an enormous "permissiveness" and was able to present eight different MBP epitopes when the HLA restriction of long-term pro liferative TCL were examined by antibody blocking ( 1 30). Each of the cytotoxic TCL described so far was restricted by HLA-DR molecules ( 1 3 1 , 1 35). HLA-DR2, -DR4 and -DR6 were able to present 9, 9, and 5 different MBP peptides respectively to TCL ( 1 3 1). Moreover, two of the peptides (87�106 and 1 5�1 72) were presented by four and three different HLA DR molecules ( 1 3 1). When cytotoxic TCLs were analyzed, similar fre quencies of long- and short-term TCL specific for this epitope were observed in DR2 + MS patients and controls as well as iIi individuals that are DR4-positive ( 1 3 1 ; R. Martin, M. Flerlage, H. F. McFarland, unpublished results). Thus, T-cell recognition of this epitope is not restric ted by a single HLA-DR allele. Moreover, a few proliferative TCL specific for 8�102 were restricted by HLA-DQ I (1 25), and it will be interesting to determine whether these differ in function from those restricted by HLA-DR, since HLA-DQ molecules are more closely associated with MS. The restriction of MBP-specific proliferative or cytotoxic T cells was further dissected in two recent studies ( 1 34, 1 39). Both were interested in which of the concomitantly expressed polymorphic DR2f3 chains (DR2a or DR2b) was the restriction element for HLA-DR2-restricted MBP specific T-cell responses. Similar to a DR2-restricted malaria cir cumsporozoite antigen ( 1 40), most of the cytotoxic and proliferative MBP specific TCL were restricted by DR2a ( 1 34, 1 39). However, TCL restricted by DR2b and specific for peptides 80-99, l 48�1 62 ( 1 34), and 1 5�172 ( 1 39) were derived from MS patients but not from controls. This is of interest insofar as MY-specific CTL from MS patients were also frequently DR2b-restricted ( 1 4 1 ). These observations raise the possibility that restric tion of certain MBP epitopes and of one or more epitopes of measles virus by HLA DR2b is unique to MS. TCR USAGE BY MBP-SPECIFIC T CELLS The expressed TCR chains in small numbers of cells derived from a TCL have been examined in a number of laboratories. In most of the studies, RNA was isolated from MBP-specific T-cell cultures and reverse transcribed before TCR cDNA was then ampli fied by PCR using either TCR family-specific primer, consensus primer, or the one-sided or anchor PCR. The observations that encephalitogenic


1 75

TCL from both PL mice and Le rats used the same TCR V/3 chains (62), despite the fact that they recognized different epitopes, raised hope that restricted TCR usage could be found in MS patients, even though the specificities and restriction elements of MBP-specific TCLs are hetero geneous. Accordingly, the observation that 84--1 02-specific TeL pre dominantly express Vf31 7 and Vf3 1 2 raised great interest ( 1 42). In addition to these two V/3 chains, a number of other V/3 elements were also found,


but to a lesser extent. These data were heavily influenced by results from one patient from whom 3 1 of 5 1 TeL had been derived. In a second study, the TCR Vf3 chain usage was analyzed in 34 MBP-specific T-cell clones from five MS patients. Restricted TCR f3-chain usage was observed in each patient, but it varied between different patients ( 1 43). The latter study is, however, difficult to compare to the first one since only limited data are available on specificity and restriction of T-cell clones. None of the clones was specific for a peptide similar to 84-- 1 02 used in the former study ( 1 4 2). A third group reported that the TCR usage was skewed towards Vf3S.2 and 6. 1 in 50 MBP-specific T-cell clones isolated from DR2 + MS patients (A. Vandenbark, personal communication). The clones had different anti gen specificities. However, here again, there is no agreement because, if the data from the first two reports (142, 1 43) are pooled, only 6 out of 83 T-cell lines or clones expressed V/35 or V/36. TCL specific for epitope 871 06 thus far have not shown restricted V f3 chain usage, although the numbers are still too small to allow definite conclusions (135; R. Martin, V. Vtz, J. E. Coligan, J. R. Richert, M. Flerlage, E. Robinson, R. Stone, W. E. Biddison, D. E. McFarlin, H. F. McFarland, unpublished results). When southern blotting was used to study the TCR f3-chain rearrange ments 1 2 different patterns were observed in 1 7 T-cell clones specific for M BP-peptide 1 50- 1 70 that were restricted by DRw 1 3 and derived from one MS patient ( 1 44). In preliminary investigations analyzing the TCRoe chain expression of MBP-specific TCL from MS patients, two laboratories found a strikingly high number of lines that express either V 0:3. 1 or V0:8.2 together with a so-far-unassigned Joe segment, JoeX ( 1 45, 1 46). Altogether, there is little agreement among the studies of TeR usage by M BP-specific T cells derived from the blood of MS patients. This work is compromised by the relatively small sample sizes and variations among patients with respect to disease type and activity; however, it is difficult to explain why there is marked variation in the results and why those investigations that have observed restricted TCR usage also find that this involves different V f3 elements. TCR USAGE IN THE CNS

A complementary approach to assessing TCR

usage by antigen-specific TCL has been to extract TCR mRNA from the


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CNS of patients with MS. Based on the assumption that T cells within infiltrates of the brain are disease-related, the usage of Va chain segments in MS brains was examined ( 1 47). Only two to four rearranged TCR Va transcripts were detected in three MS brains indicating that lymphocytes in the brain used only a limited number of TCR a chains. Val 0 was rearranged in plaques from all three MS brains, Va8 and Va1 2 were rearranged in two brains ( 147). When the junctional regions of 25 cDNA clones from MS plaques were analyzed, only two different sequences were found. No Va chain expression was seen in control brains ( 1 47). In a larger subsequent study, Vf3 rearrangements and molecular HLA typing were performed ( 148). In the entire patient population, limited heterogeneity of Va and Vf3 chain rearrangements was observed. However, when the pa tients were stratified according to HLA background 7/7 individuals that were HLA-DRB I 1 50 1 , DQAl 0 1 02, DQBl 0602 expressed Vf35.2 tran scripts and 6/7 expressed Vf36 in brain white matter plaques ( 1 48). From this data and the above mentioned expression of Vf35.2 and Vf36 by MBP specific T-cell clones, it is postulated that MBP-specific T cells might be involved in the pathogenesis of MS (A. Vandenbark, personal com munication; 1 48). To interpret TCR expression in the brain, additional information is required. In particular, it will be interesting to correlate the TCR expression in the brain and the blood of the same individual. Furthermore, it will be important to determine whether encephalitogenic T cells expressing Vf38.2 can be demonstrated in brain tissue ofPL mice and Le rats and to examine the extent to which other TCR are expressed. How T cells expressing yo TCR heterodimers that have been shown in MS lesions contribute to the disease process is also not yet clear (80, 1 49).

Evidence of Environmental Factors It is widely believed that the immune-mediated process, discussed above is triggered by an environmental factor(s). Evidence of this is derived from epidemiological studies. The geographic distribution ofMS is not uniform, and the prevalence is higher in regions with cooler climates of both hemi spheres. Furthermore, individuals that migrate before the age of 1 5 acquire the risk of getting MS that is found in the geographical region to which they migrate ( 1 50). Individuals that migrate after age 1 5, carry the risk of developing the disease that is seen in the geographic region of origin. In addition, "epidemics" of MS have been described. The most extensively studied is the occurrence of MS on the Faroe islands where there were no cases of MS prior to 1 940. Subsequent to the occupation of the islands by the British troops during World War II the disease appeared. Analysis of the Faroe islands cases suggests that MS is related to a widespread infection



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transmitted between age 1 3-26, and that three separate epidemics have occurred ( 1 5 1 ). Similar clusters of disease have been reported from other areas, but these were evaluated in less detail ( 1 50). The epidemiological data indicate that MS is related to an infectious agent encountered during childhood. There has been extensive search for a possible causative agent. Over the years at least 1 2 different viruses ranging from paramyxo- to retroviruses have been implicated in the etiology of MS, but thus far, no single agent related to the disease has been 'confirmed ( 13). It is clear from the study of both animal and human disease that MBP specific T cells can be activated during infections. MBP-specific lympho cytes have been demonstrated in the blood during postinfectious enceph alomyelitis after measles infection as well as in the CSF during Lyme radiculomyelitis and rubella panencephalitis ( 1 3, 1 9, 1 52, 1 53). Furthermore, experimental infections with measles or coronavirus stimulate MBP-speci fic T cells which can subsequently transfer an EAE-like disorder (26, 27). The presence of MBP-specific T cells in the blood of healthy controls shows that cells specific to CNS autoantigens belong to the "normal" T cell repertoire, and it is possible that these cells are expanded by myelin release during MS ( 154) or virus infection. Alternatively, self-reactive T cells could be activated by the recognition of epitopes shared by virus and autoantigen. Sequence homology between myelin proteins and a number of viruses has been shown (1 55, 1 56), but T cells that recognize both viral and myelin epitopes have not yet been demonstrated in vitro.

Current Research Strategies Advances in the understanding of EAE over the past decade have provided the background for a considerable amount of research in MS. There are at least three possible conclusions. 1 . The most pessimistic is that none of the new information derived from the study of EAE is applicable to MS. One could speculate as to why this is the case; for example, MS could be secondary to latent infection by an as-yet-unknown infectious agent. However, it seems premature to leap to this possibility. 2. The most optimistic conclusion is that the observation made in inbred species in EAE will hold in MS, and immunotherapies such as vaccination with T cell receptor peptides will provide an effective treatment. However, the existing data don't support this. Although dominant epitopes of MBP have been detected, it is not clear whether these are disease-specific. Some studies do indicate potentially exciting skewing of TCR usage, but domi nant TCR usage also has not been established. There are two experimental approaches: (a) Perform additional experiments in search of data that support the premise that MS is like EAE. (b) Assume that the principles T-CELL SPECIFICITY AND FUNCTION


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established in EAE are valid in MS and proceed with the next step. Based on the restricted usage of V[3S.2 and studies of EAE, the group led by Vandenbark, Offner et al recently introduced a vaccination study in a small group of MS patients that will receive a peptide homologue to the CDR2 region of V[3S.2 (A. Vandenbark, personal communication). If this audacious approach shows a meaningful effect, even in a few patients, it would establish that immunological processes were occurring and would provide rationale for greater effort. 3. A possibility that has support, at least theoretically, is that immuno logical processes responsible for EAE are operative in MS, but that in the overall population of patients there are a variety of immune responses directed at multiple target antigens instead of a single immunodominant epitope. Each of these could be influenced by individual HLA and TCR genes. The rationale for this point of view is derived from the following facts: (a) Humans are highly outbred. (b) The human HLA system is considerably more diverse than the MHC of rodents. (c) Even in inbred mice, both MBP and PLP are encephalitogenic, and each protein contains at least two encephalitogenic epitopes. In individual patients immune reactivity against a single dominant epitope could produce disease; hence the experimental vaccination procedures currently in progress are relevant. In addition, approaches that produce downregulation of the immune response such as IFN-[3 and TGF-f3 could be effective in an immunological process directed at multiple epitopes . LOCAL IMMUNE REACTIVITY IN THE CNS In comparison to other tissues, the CNS is immunologically unique. This is in part due to the separation from the systemic circulation by the BBB ( 1 57) which is largely formed by tightjunctions of the brain capillary endothelium. This impedes the passage of macromolecules and cells into the CNS. A second major difference is the low or undetectable expression of HLA molecules in the CNS ( 158), and there are indications that HLA expression is regulated differently in CNS cells than in other tissues (1 59). These special features of the CNS have implications to the pathogenesis of MS. As mentioned above, recent MRI findings indicate that an early sign of lesion development is break down of the BBB, which can be visualized by leakage of paramagnetic substances into the parenchyma (72). The events that lead to increased permeability of the BBB and to the migration of inflammatory cells into the tissue are poorly understood. Histopathological studies and in vitro investigations have shown that lymphocyte-endothelial cell interactions occur ( 1 60). IFN-y can induce the expression of MHC--class II molecules not only on brain capillary endothelial cells, but also on astro- and microglial cells ( 1 6 1-163). Sub-



1 79

sequently, these cells can present antigens to T cells ( 1 64-166). These observations have potential implications to both physiological and patho logical immune responses in the CNS; however, evidence that such events occur in vivo is lacking, and other components such as adhesion molecules may be more important ( 167). Another cytokine, TNFa, activates endo thelial cells ( 1 68) and causes demyelination in vitro (1 69). The potential importance of TNF is further underlined by the observation that enceph alitogenicity correlates with production of TNFa and -/3 (lymphotoxin) ( 1 70) and that an antibody against TNF protects animals from EAE ( 1 7 1). Further characterization of the interactions between immune cells and components of the CNS is clearly indicated. The study of the tissue-specific expression of HLA-molecules, lymphokines, and adhesion molecules at the molecular level should provide insight into the early events involved in lesion development and possibly provide rationales for therapeutic intervention. ACKNOWLEDGMENTS We thank Sherri Davenport and Millie Fenton for preparation of the manuscript. Literature Cited

1 . Adams, R. D., Kubik, C. S. 1952. The morbid anatomy of the demyelinative diseases. Am. 1. Med. 12: 5 1 0-46 2. Peters, A. 1960. The structure of myelin sheaths in the central nervous system of xenopus laevis (Daudin). J. Biophy. Biochem. Cytol. 7: 1 21-26 3. Green, B. G. 1954. The formation from the Schwann cell surface of myelin in peripheral nerves of chick embryos. Exp. Cell Res. 7: 558-62 4. Bunge, M. B., Bunge, R. P., Pappas, G. D. 1 962. Electron microscopic demonstrations of connection between glia and myelin sheaths in the develop ment mammalian brain. J. Cell Bioi. 1 2: 448-53 5. Waxman, S. G. 1 985. Structure and function of the myelinated fiber. In Handbook of Clinical Neurology, De myelinating Diseases, ed. J. C. Koet

sier, 3(47): 1-28. Amsterdam/New York: Elsevier Sci. 6. Quarles, R. H., Morell, P., McFarlin, D. E. 1989. Diseases involving myelin. In Basic Neurochemistry, ed. G. Siegel, B. Agranoff, R. W. Albers, P. Molinoff, pp. 697-713. New York: Raven. 4th ed. 7. Adams, C. W. 1 989. A colour atlas of

8.

9.

10.

11.

1 2.

13.

multiple sclerosis and other myelin dis orders. London: Wolfe Med. Suzuki , K. 1989. Genetic disorders of lipid, glycoprotein, and mucopoly saccharide metabolism. In Basic Neuro chemistry, ed. G. Siegel, B. Agranoff, R. W. Albers, P. Molinoff, pp. 7 1 5-32. New Yark: Raven. 4th ed. Moser, H. W., Moser, A. E., Singh, I., O'Neil, B. P. 1984. Adrenoleuko dys trophy-Survey of 303 cases: bio chemistry, diagnosis and therapy. Ann. Neural. 16: 628--41 Bernheimer, H., Budka, H., Muller, P. 1 983. Brain tissue immunoglobulins in adrenoleukodystrophy: a comparison with multiple sclerosis and systemic lupus erythematosus. Acta Neuropath. 59: 95-102 Ludwin, S. K. 1978. CNS demy elination and remyelination in the mouse. An ultrastructural study of cuprizone toxicity. Lab. Invest. 39: 597612 Smith, M . L., Benjamin, J . A . 1984. Model systems for the perturbations of myelin metabolism. In Myelin, ed. P. Morell, pp. 441-87. New York: Plenum John son R. T. 1985. Viral aspects of ,

1 80


multiple sclerosis. In Handbook of

Clinical Neurology, Demyelinating Dis eases, ed. J. C. Koetsier, 3(47): 3 1 936. Amsterdam/New York: Elsevier

14.

Sci. Levy, R. M., Bredeson, D. E., Rosen blum, M. L. 1 988. Opportunistic cen


tral nervous system pathology in pa

tients with AIDS. Ann. Neurol. 23 (Suppl. I): 7-1 2 1 5 . Vernant, 1. c . , Maurs, L . , Gessain, A., Barin, F., Gout, 0., Delaporte, J. M . , Sanhadji, K . , Buisson, G. 1987. En demic tropical spastic paraparesis associated with HTLV-I: a clinical and seroepidemiological study of 25 cases. 1 6.

17.

Ann. Neurol. 2 1 : 1 23-30

Osame, M., Matsumoto, M., Usuku, K. 1987. Chronic-progressive myel opathy associated with elevated anti bodies to human T-1ymphotropic virus type 1 and adult T-cell leukemia-like cells. Ann.

Neurol. 2 1 : 1 1 7-22

Jacobson, S., Gupta, A., Mattson, D. H., Mingioli, E. S., McFarlin, D. E. 1 990. Immunological studies in tropical spastic paraparesis. Ann. Neurnl. 27: 149-56

Jacobson, S., Shida, H., McFarlin, D. E., Fauci, A. S., Koenig, S. 1990. Cir culating CD8 + cytotoxic T lympho cytes specific for HTLV-I pX in pa tients with HTLV-I associated neuro logical disease. Nature 348: 245-48 1 9. Johnson, R. T., Griffin, D. E., Hirsch, J. S., Wolinsky, J. S., Rodenbeck, S., Lindo De Soriano, I., Vaisberg, A. 1984. Measles encephalomyelitis clini cal and immunological studies. N. Engl. 18.

25. Yamada, M., Zurbriggen, A., Fuji nami, R. S. 1 990. Monoclonal antibody to Theiler's murine encephalomyelitis virus defines a determinant on myelin and oligodendrocytes, and augments demyelination in experimental allergic encephalomyelitis. J. Exp. Med. 1 7 1 : 1893-1907

Watanabe, R., Wege, H., ter Meulen, V. 1 983. Adoptive transfer of EAE-like lesions from rats with coronavirus-in duced demyelinating encephalomyeli tis. Nature 305: 1 50-52 27. Liebert, U. G., Linington, C., ter Meulen, V. 1 978. Induction of auto immune reactions to myelin basic pro tein in measles virus encephalitis in Lewis rats. J. Neuroimmunol. 1 7 : 10326.

36

Narayan, 0., Sheffer, D., Clements, J. E. 1 985. Restricted replication of lenti viruses. J. Exp. Med. 1 62: 1 954-69 22. Chamorro, M., Aubert, c., Brahic, M. 1 986. Demyelinating lesions due to Theiler's virus are associated with cen tral nervous system infection. J. Virol.

21.

23.

Allergic Encephalomyelitis: A Useful Modelfor Multiple Sclerosis, ed. E. C. Alvord, Jr., pp. 146-5 1 1 . New York:

920-27

24.

33.

Lipton, H. L., Dal Canto, M. C. 1 976. Theiler's virus induced demyelination prevention by immunosuppression. Science 1 92: 62--64

Liss Kamholz, J., de Ferra, F., Puckett, C., Lazzarini, R. 1986. Identification of three forms of human myelin basic pro tein by cDNA cloning. Proc. Natl.

Acad. Sci. USA 83: 4962--66

34.

Fritz, R. B., Skeen, M. J., Chou, C.-H. J., Zamvil, S. S. 1990. Localization of an encephalitogenic epitope for the SJL mouse in the N-terminal region of myelin basic protein. J. Neuroimmunol.

35.

Tuohy, V. K., Sobel, R. A., Lees, M. B. 1 988. Myelin proteolipid protein induced experimental allergic ence phalomyelitis: variations of disease expression in different strains of mice.

57: 992-97

Clatch, R. J., Lipton, H. L., Miller, S. D. 1986. Characterization of Theiler's virus murine encephalomyelitis (TMEV)-specific delayed-type hyper sensitivity responses in TMEV-induced demyelinating diseases: correlation with clinical signs. J. Immunol. 1 36:

58: 39-53

Fritz, R., McFarlin, D. E. 1 989. Ence phalitogenic epitopes of myelin basic protein. In Chem. Immunol., ed. E. Ser carz, 46: 1 01-25. Basel: Karger 3 1 . Kabat, E. A., Wolf, A., Bezer, A. E. 1 946. The rapid production of acute disseminated encephalomyelitis in rhe sus monkeys by injection of brain tissue with adjuvants. Science 1 04: 362--63 32. Martenson, R. E. 1 984. Myelin basic protein speciation. In Experimental 30.

J. Med. 3 10: 1 3 7-41 20. Haase, A. T. 1 986.

Pathogenesis of lentivirus infections. Nature 322: 1 30-

18

Remlinger, J. 1905. Accidents para Iytiques au cours du traitment anti rabique. Ann. Inst. Pasteur 1 9 : 625-46 29. Rivers, T. M . , Sprunt, D. H., Berry, G . P. 1 933. Observations o n attempts to produce acute disseminated ence phalomyelitis in monkeys. J. Exp . Med.

28.

36.

26: 239-43

J. lmmunol. 140: 1 868-73

Tuohy, V. K., Lu, Z., Sobel, R. A., Laursen, R. A., Lees, M . B. 1989. Identification of an encephalitogenic determinant of myelin proteolipid pro-


37.


38.

39.

40.

41.

42.

43.

tein for SJL mice. J. Immunol. 142: 1 523--27 Quarles, R. H. 1 988. Myelin-associated glycoprotein: functional and clinical aspects. In Neurological Research, Vol. II: Neuronal and Glial Proteins Struc tures, Function, and Clinical Appli cation, ed. P. J. Marangos, I. Campbell, R. M. Cohen, pp. 295-320. New York: Academic Le Bar, R., Lubetski, c., Vincent, C., Lombrail, P., Boutry, J. M . 1986. The M2 autoantigen of central nervous sys tem myelin, a glycoprotein present in oligodendrocyte membrane. CUn. Exp. lmmunol. 66: 423-43 Cohen, I. R. 1 98 1 . Multiple sclerosis" like disease induced in rabbits by immunization with brain gang1iosides. Isr. J. Med. Sci. 1 7 : 7 1 1-14 Levine, S., Wenk, E. J. 1965. A hyper acute form of allergic encephalo myeliti s . Am. J. Pathol. 47: 61-88 Bernard, C. C. A., Carnegie, P. R. 1 973. Experimental allergic encephalomyeli tis in mice: immune response to mouse spinal cord and myelin basic protein. J. Immunol. 1 1 4: 1 5 37-40 Raine, C. S. 1983. Multiple sclerosis and chronic relapsing EAE: com parative ultrastructural neuropathol ogy. In Multiple Sclerosis, ed. 1. F. Hall pike, C. W. M. Adams, W. W. Tour tellotte, pp. 4 1 3-78. Baltimore: Wil liams & Wilkins Moore, G. W. R., Traugott, D., Farooq, M., Norton, W. T., Raine, C. S. 1984. Experimental autoimmune encephalomyelitis. A generation of de myelination by different myelin lipids.

49.

50.

51.

52.

53.

54.

Lab. Invest. 5 1 : 41 6-24

44. Paterson, P. Y. 1 960. Transfer of allergic encephalomyelitis in rats by means of lymph node cells. J. Exp . Med. 1 1 1 : 1 1 9-33 45. Stone, S. H. 1 96 1 . Transfer of allergic encephalomyelitis by lymph node cells in inbred guinea pigs. Science 1 34: 6 1 921 46. Panitch, H . S., McFarlin, D . E. 1977. Experimental allergic encephalomye litis: enhancement of cell-mediated trans fer by concanavalin A J. Immwrol. 1 19: 1 1 34-37 47. Richert, J. R., Driscoll, B. G., Kies, M . W . , Alvord, E. C. Jr. 1979. Adoptive transfer of experimental allergic ence phalomyelitis: incubation of rat spleen cells with specific antigen. J. Immunol. 1 22: 494-96 48. Ben-Nun, A., Wekerle, H., Cohen, I. R. 1 98 1 . The rapid isolation of clonable antigen-specific T lymphocyte lines

55.

56.

57.

58.

181

capable of mediating autoimmune en cephalomyelitis. Eur. J. Immunol. 1 1 : 1 95-99 Pettinelli, C. B., McFarlin, D. E. 1 9 8 1 . Adoptive transfer of experimental allergic encephalomyelitis in SJL/J mice after in vivo activation of lymph node cells by myelin basic protein: requirement for Lyt-1 + 2 - T lym phocytes. 1. Immunol. 127: 1 420-23 Steinman, L., Rosenbaum, 1., Sriram, S., McDevitt, H. 0. 1 98 1 . In vivo effects of antibodies to immune response gene products: Prevention of experimental allergic encephalomyelitis. Froc. Natl. A cad. Sci. USA 78: 71 1 1-14 Aharoni, R., Teitelbaum, D., Arnon, R., Purl, J. 199 1 . Immunomodulation of experimental allergic encephalitis by antibodies to antigen Ia complex. Nature 35 1 : 147-50 Sakai, K., Zamvil, S. S., Mitchell, D.

J., Hodgkinson, S., Rothbard, J. 8.,

Steinman, L. 1989. Prevention of experimental encephalomyelitis with peptides that block interaction of T cells with major histocompatibility complex proteins. Froc. Natl. A cad. Sci. USA 86: 9470-74 Wraith, D. C., Smilek, D. E., Mitchell, D. J., Steinman, L., McDevitt, H . o. 1989. Antigen recognition in auto immune encephalomyelitis and the potential for peptide-mediated im munotherapy. Cell 59: 247-55 Lamont, A G., Sette, A, Fujinami, R., Colon, S. M., Miles, c., G rey, H. M. 1 990. Inhibition of experimental auto immune encephalomyelitis induction in SJL/J mice by using a peptide with high affinity for Ia molecules. J. Immunol. 145: 1 687-93 Teitelbaum, D., Aharoni, R., Arnon, R., Sela, M . 1 988. Specific inhibition of the T-cell response to myelin basic protein by the synthetic copolymer Cop- 1 . Proc. Nat!. Acad. Sci. USA 85: 9724-28 Brostoff, S. W., Mason, D. W. 1 984. Experimental allergic encephalomye litis: successful treatment in vivo with a monoclonal antibody that recognizes T helper cells. J. Immunol. 1 33: 1 93842 Waldor, M. K., Sriram, S., Hardy, R., Herzenberg, L. A., Herzenberg, L. A., Lanier, L., Lim, M., Steinman, L. 1 985. Reversal of experimental allergic ence phalomyelitis with a monoclonal anti body to a T cell subset marker (L3T4). Science 227: 4 1 5-17 Kennedy, M . K., Tan, L.-J., Dal Canto, M. C., Miller, S. D. 1 990. Regu-

1 82


lation of the effector stages of experi mental autoimmune encephalomyelitis via neuroantigen-specific tolerance in duction. J. Immunol. 145: 1 1 7-26 59. Zamvil, S. S., Steinman, L. 1 990. The T lymphocyte in experimental allergic encephalomyelitis. Annu. Rev. Immu nolo 8: 579-62 1


60.

Acha-Orbea, H., Mitchell, D. J., Tim mermann, L., Wraith, D. c., Tausch, G. S. 1 988. Limited heterogeneity of T cell receptors from lymphocytes medi ating autoimmune encephalomyelitis allows specific immune intervention. Cell 54: 263-73

Urban, J. L., Kumar, V., Kono, D. H., Gomez, c., Horvath, S. J., Clayton, J., Ando, D. L., Sercarz, E. E., Hood, L. 1 988. Restricted use of the T cell recep tor V genes in murine autoimmune encephalomyelitis raises possibilities for antibody therapy. Cel/ 54: 577-92 62. Ben-Nun, A., Wekerle, H., Cohen, I. R. 198 1 . Vaccination against autoimmune encephalomyelitis with T-lymphocyte line cells reactive against myelin basic protein. Nature 293: 60-61 63. Burns, F. R., Li, X., Shen, N., Offner, H., Chou, Y. K., Vandenbark, A., Heber-Katz, E. 1 989. Both rat and mouse T cell receptors specific for the encephalitogenic determinant of myelin basic protein use similar Vrx and Vf3 chain genes even though the major histocompatibility complex and ence phalitogenic determinants being recog nized are different. J. Exp. Med. 1 69:

61.

27-39

64.

Vandenbark, A. A., Hashim, G., Offner, H. 1989. Immunization with a synthetic T-cell receptor V-region pep tide protects against experimental autoimmune encephalomyelitis. Na

65.

Offner, H., Hashim, G. A., Vanden bark, A. A. 1 990. T cell receptor pep tide therapy triggers autoregulation of experimental encephalomyelitis. Sci

administration of myelin basic protein. Higgins, P. 1., Weiner, H. L. 1 988. Sup pression of experimental autoimmune encephalomyelitis by oral adminis tration of myelin basic protein and its fragments. J. Immunol. 140: 44

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