The T lymphocyte in experimental allergic encephalomyelitis.

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Annu. Rev. Immunol. 1990.8:579-621 Copyright © 1990 by Annual Reviews Inc. All rights reserved

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THE T LYMPHOCYTE IN

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EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS Scott S. Zamvil and Lawrence Steinman Departments of Neurology, Pediatrics and Genetics, Stanford University School of Medicine, Stanford, California 94305 KEY WORDS:

autoimmunity, T cell receptor, experimental allergic encephalo myelitis, immunotherapy, major histocompatibility complex.

INTRODUCTION Discrimination between foreign and self antigens is necessary for normal immune function. Whereas exposure to foreign antigens can lead to their elimination and subsequent specific immunity, an unregulated immune response to self antigens may culminate in autoimmune disease. Experi mental allergic encephalomyelitis (EAE) is a prototype for antigen-specific T cell-mediated autoimmune disease (1, 2). The auto antigen in EAE is myelin basic protein (MBP), the predominant protein of central nervous system myelin. EAE is mediated by class II-restricted, MBP-specific CD4+ T lymphocytes. Certain forms of EAE are characterized by relaps ing paralysis, with histopathology demonstrating perivascular lymphocytic infiltrates and demyelination within the central nervous system (eNS). Because of these and other clinical and histologic features, relapsing EAE is considered as a model for the human demyelinating disease multiple sclerosis (MS) (3, 4). Furthermore, susceptibility for both EAE and MS (5) is linked to class-II (immune response) genes. As a model for human autoimmune disease, EAE has been useful for testing novel forms of immunotherapy. A major goal for such testing is to develop modalities that can selectively inactivate or eliminate only those cells involved in disease pathogenesis. Several studies have demonstrated that antibodies targeted at class-II molecules, CD4, and T cell receptor

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(TCR) gene products as well as other molecules involved in immune regulation are effective in preventing or treating EAE. In this article we review the field of EAE, focusing on the identification of the autoantigen MBP and the T lymphocyte as the mediator of EAE. Experimental evidence demonstrating the relationship between the MHC and susceptibility to EAE is described as well as work examining T-cell receptor gene expression in the encephalitogenic response to MBP. A description of various approaches to immune intervention in EAE is


included. Although EAE is a prototype for antigen-induced, T-celI mediated autoimmune disease, it is considered by many to be a good model for MS, a spontaneous demyelinating disease in humans. The similarities and differences between EAE and MS are discussed.

T-CELL RECOGNITION The immune system of a given host can recognize a multitude of different antigens; the response to each antigen, however, is specific. To understand how lymphocytes participate in the pathogenesis of autoimmune disease requires a firm understanding of antigen recognition. The two separate lineages of lymphocytes, T cells and B cells, achieve their respective speci ficities in different ways. Whereas B cells recognize soluble antigen via their immunoglobulin receptor in a direct binary interaction, T cells in general recognize nominal antigen only in association with a product of the MHC, the class-I and class-II antigens. T-cell recognition, therefore, involves a ternary interaction, requiring antigen, MHC, and the clonaIJy distributed antigen-specific T cell receptor (TCR) (6). CD4+ (TH) cells recognize antigen in association with class-II molecules expressed on anti gen presenting cells (APC). CD8 + (Td cells recognize foreign antigen in association with class-I molecules. The ternary interaction of antigen, MHC, and TCR is also referred to as the trimolecular complex. The molecular nature of this complex has not been elucidated. However, evi dence supports binary interaction of individual components (7-12).

The Polymorphic MHC The genes of the MHC are located on chromosome six in humans and chromosome 17 in mice. Class-I molecules are cell-surface proteins ex pressed by all nucleated cells within an individual. In humans these are the HLA-A, B, and C antigens (13). The homologous proteins in mice are the H-2 K, D, and L antigens. Class-l molecules are composed of two polypeptide chains, a polymorphic 45-kilodalton (kd) protein that is non covalently linked to PTmicroglobulin, a 12-kd monomorphic protein. Crystallographic studies indicate that alpha helixes within the al and a2


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domains fold t o form the antigen binding cleft, with a majority o f the polymorphic residues surrounding this cleft ( 1 4, 1 5). Recent studies sup port the direct binding of class I with antigenic peptides recognized by Tc cells ( 1 6). Class-II molecules are cell-surface glycoproteins expressed constitutively by macrophages, B-cells, and dendritic cells. The class-II molecules in humans are the HLA-DP, DQ, and DR antigens. There are two isotypic forms in mice, I-A and I-E. I-A is the homolog of HLA-DQ, and I-E is the homolog of HLA-DR ( 1 7). These cell-surface glycoproteins are heterodimers, composed of a 34-kd oc-chain and a 28-kd f3-chain. Based on the crystallographic studies of class I, a similar model has been proposed for class II. In this model the polymorphic residues of the oc l and f3 l domains fold to create a single antigen binding site (29). Recent studies have demonstrated a direct, although low affinity, binding between MHC class-II molecules and antigenic peptides (7- 1 2). The expression of the polymorphic MHC molecule is of central impor tance to the normal and autoimmune T-cell response. Only those peptide antigens that bind a specific allelic MHC molecule are presented to T cells. In other words, the MHC composition of an individual controls which portiones) of an antigen are selected for presentation to T cells. This concept is called "determinant selection" ( 1 8). Recent studies demonstrate that the expression of MHC can influence the potential T-cell repertoire, by both positive and negative selection ( 1 9, 20). Both qualitative and quantitative changes in expression of class-II genes influence the T-cell response to antigens and susceptibility to autoimmune conditions. For' example, the mutant mouse strain B6.C-H-2bm 1 2 (bm 1 2) differs from its parental strain C57blj6 (B6) only at residues 68, 7 1 , and 72 within the first domain of the I-Af3 chain (2 1). Although bm l 2 shares the same genetic background, its response to certain antigens differs from that of the par ental B6 strain. For instance, B6 mice respond to beef insulin, but not sheep insulin. These two antigenic proteins are identical except at one residue, amino acid 9. In bm 1 2, the response is just the opposite; bm 1 2 responds to sheep insulin, but not beef insulin (22). Thus, a n isolated change in the I-A restricting element can have a profound influence on the T-cell response to similar antigenic protein molecules. Furthermore, whereas B6 mice develop experimental autoimmune myasthenia gravis (EAMG) following immunization with acetylcholine receptor, bm 1 2 is resistant to EAMG (23). However, this difference in susceptibility to EAMG of bm 1 2 mice may reflect not only the influence of polymorphic differences within the I-A molecule but quantitative differences in the level of I-A expression in bm 1 2 mice (24). The association of human autoimmune disease and HLA is well estab lished (25). For many of these diseases, including insulin dependent dia-


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betes mellitus (IDDM), rheumatoid arthritis, thyroiditis, SLE, myasthenia gravis and MS, as well as with other diseases, the strongest association occurs with specific allelic class-II (HLA-D) genes. This association with class II provides indirect evidence that T cells are involved in these auto immune conditions. Human susceptibility to two diseases, IDDM and pemphigus vulgaris, has been linked to a polymorphism at residue 57 in the DQ f3 chain (26). Thus, in diabetes, alanine, valine, or serine at position 57 is associated with increased susceptibility, while Asp at position 57 correlates with resistance. That amino acid 57 is thought to he located in the antigen-binding cleft of class-II molecules (29) suggests that residue 57 may participate directly in Ag-MHC interaction.

The Antigen and T-Cell Epitopes B cells recognize conformational determinants of protein antigens via their immunoglobulin receptor molecules. These epitopes, dependent upon tertiary structure, are usually located on the hydrophilic surfaces of proteins. In contrast, T cells recognize contiguous stretches of amino acids within protein molecules. These peptide fragments, which are generated by proteolytic cleavage, bind to specific MHC molecules. In vitro studies have demonstrated that the minimum peptide length that can initiate a T cell response is 7-8 residues, although peptides > 1 0 residues are presented more efficiently (27). The length of peptides presented to T cells.in vivo is not known. Direct binding assays with solubilized class-II molecules made it evident that peptides bound class II with different affinities and that the poly morphisms within class II molecules contribute to thes(� differences in affinity. These results, demonstrating increased relative affinity for the binding of immunogenic peptides to appropriate class II, provide further evidence for determinant selection in the T-cell response. In vitro com petition studies suggested that although any class-II molecule can bind a variety of different peptides, there appears to be a single antigen binding site ( 1 0), not multiple sites as suggested earlier from antibody blocking studies (28). The single binding site is in agreement with the model for class II (29), based on the crystallographic studies of class-I molecules ( 1 4, 1 5). Presentation of antigen to TH cells requires antigen processing by class II-bearing cells. The end result of processing is the binding and exposure of individual epitopes within proteins. Antigen processing involves proteo lytic cleavage of antigen (and/or denaturation) (30, 3 1 ) . Using glutar aldehyde-fixed, antigen-presenting macrophages, Shimonkevitz et at (32) demonstrated that T-cell hybridomas specific for ovalbumin responded to proteolytic digests of ovalbumin, but not to intact or denatured ovalbumin.



583

The lysosomatropic agents chloroquine and ammonium chloride which inhibit proteases, as well as the protease inhibitor leupeptin, have been shown to inhibit presentation of intact antigen but not enzymatic digests (30-33). For the antigen myoglobin, Streicher et al (33) demonstrated that denatured but not native myoglobin could be presented to myoglobin specific T-cell clones by APC treated with lysosomal inhibitors. Perhaps the most convincing evidence for antigen processing has come from the studies by Watts et al (34) who demonstrated that planar lipid membranes containing class-II molecules could present peptide digests of ovalbumin but not intact "native" ovalbumin to T-cell hybridomas specific for ovalbumin. Common structural characteristics among T-cell epitopes have emerged from the comparison of a large number of determinants. Delisi & Berzof sky (35) have suggested that peptides which form an alpha helical con formation are more likely to be recognized by T cells. Rothbard (36) has found empirically that a majority of T-cell epitopes contain a common motif. This template includes a core of four or five contiguous amino acids, a charged residue or glycine, followed by two or three hydrophobic residues, and in the fourth (or fifth) position, a charged or polar amino acid. Although neither model alone accounts for all T-cell epitopes, these models have proven very useful in predicting epitopes of foreign and autoantigens (37-39). A single antigen may have multiple overlapping or nonoverlapping T cell epitopes. However, not all pep tides that bind to MHC may be recog nized by T cells with equal efficiency. For example, there are cases in which a T-cell response to a specific epitope is elicited when an individual is immunized with a known immunogenic peptide, but not when immunized with the intact protein containing the same sequence (40, 4 1). A number of factors may influence whether individual peptides are recognized in vivo. Some suggest that there is a hierarchy in the capacity of individual immunogenic epitopes within a protein to stimulate T cells (42). This dominance may reflect competition among individual peptides for a given class II-restricting element. Different requirements may exist for antigen presentation with preferential sites for proteolytic cleavage. Furthermore, certain protein determinants may act as suppressor epitopes, influencing T-cell responsiveness to separate distant determinants within the same protein (40-42). Finally, the response to individual epitopes may be influ enced by the available T-cell-receptor repertoire ( 1 9 , 20, 43).

The T Cell A ntigen Receptor The clonally distributed antigen-specific receptor on T cells is a trans membrane heterodimer composed of a 45-kd a-chain and 43-kd J3-chain


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in association with the CD3 complex (44-49). Like immunoglobulins, the rx and [3 chains of the TCR each contain a variable domain, involved in recognition, and a constant region transmembrane "anchor" domain (50). The oc-chain is encoded on chromosome 14 in both human and mouse; the [3-chain is encoded on chromosome 7 in humans and 6 in mice (50, 5 1) . Similar t o the organization o f immunoglobulin genes, the genes encoding the rx and [3 chains of the TCR are discrete pools of noncontiguously encoded variable (V), diversity (D) ([3 chain only), joining (1), and constant (C) gene segments, which undergo recombination during maturation to create unique Va-la and V[3-D[3-1[3 combinations. These gene com binations are then joined to their respective constant region genes by RNA splicing following transcription (50), creating Voc-Ja-Coc and V[J-D[3-J[3-C[3 RNA molecules that are translated to mature oc and [3 chains. As a result of allelic exclusion, each T cell expresses only one TCR product (50). However, the potential T cell repertoire is extremely large. There are estimated to be approximately 1 00 germline Vrx gene segments and 50-- 1 00 Ja genes in both humans and mice. In mice there are 20-30 V[3, two D[3, 1 2 J[3 genes encoded in the germline. Based on somatic recombination ofindividual gene segments and independent combinatorial association of individual rx and [3 chain genes, it is estimated that 1 07 unique TCR molecules can be created. Further TCR heterogeneity is possible. As reviewed by Kronenberg et al (49), the following mechanisms may be employed: functional diversity There is imprecision in V[3-D[3, D[3-J[3, and Vrx-Jrx joining, allowing nucleotides on either joining end to be deleted. N-region diversity Additional random nucleotide sequences can be inserted in TCR oc and [3 chain junctional sequences. D-region diversification D[3-region genes can be translated in all three reading frames. Somatic hypermutation, which contributes additional diversification of Ig, does not play a significant role in TCR repertoire (50). Utilizing these additional mechanisms of diversification, the potential number of unique TCR molecules may be as large as 1 0 15. Several studies have investigated the relationship between TCR usage and Ag/MHC specificity. Although the potential TCR repertoire is large, in general, a restricted use of TCR gene elements has been found among T-cell clones and T-cell hybridomas that share similar antigen fine speci ficity and MHC restriction. Of the different antigen systems examined, the T-cell response to the C-terminus cytochrome c has been investigated most



585

thoroughly. Most T cells that recognize this epitope are restricted by I-E molecules. Among clones that recognize this epitope, fine differences in specificity and different alloreactivities are observed (52-55). A majority of the T-cell clones analyzed use a member of the Val l family (54, 55). Differences in fine specificity and alloreactivity correlate with TCR gene segment usage. Similar results have been obtained in other antigen systems in which T-celJ clones with similar fine specificity have been examined (5658). The structural basis for AgjMHC recognition by TCR is still obscure. A model for independent recognition of antigen and MHC is not correct; as Kappler et al showed in 1 9 8 1 , antigen and MHC specificities do not reassort upon fusion of T cells with unrelated antigen and MHC speci ficities (59). However, evidence does exist supporting the individual con tribution of V-gene products in the determination of antigen specificity, independent of MHC restriction. In the response to the hapten p-azo benzene-arsonate (Ars), a majority of T-cell clones examined, sharing the same antigen specificity but different MHC restriction, utilized the same Va gene, Va3 (56). Va3 was used in conjunction with separate Ja genes, and the expressed Va chain associated with different P chains. Furthermore, transfer of the Va 3 gene to a T cell with different antigen specificity but sharing the same restriction element conferred Ars specificity to the recipient T cell. Although the initial TCR gene sequencing failed to show any simple correlation between TCR V-gene expression and MHC restriction (60), more recent studies demonstrate that V-gene usage is influenced by MHC. In one study of the response to sperm whale myoglobin in DBA/2 (H2d) mice, most (1 0 of 1 1 ) T-cell clones (representing different myoglobin specificities) that used a member of the TCR Vp8 family were I-E restricted, whereas VPS- T cell clones were restricted by I-A (6 1). In this strain, the Vp8 subfamily is expressed by approximately 20% of mature T lymphocytes (62). In another strain, it appears that Vp8 usage may correlate with I-A restriction ( 39). The most striking result demonstrating the influence of MHC on TCR V-gene usage has come from the laboratory of Kappler & Marrack. They showed a strong correlation between expression of the VPl 7 gene segment and I-E alloreactivity in strains which express I-A, but not I-E (19, 20). In I-E+ strains and hybrid (I-E+ x 1E-) F I mice, T cells that express Vp1 7 are absent in peripheral lymphoid organs. VPI r T cells are detectable in the thymus, suggesting that these T celJs are clonally deleted in the thymus. Similar results were found for TCR VPI I expression; I-E + strains, but not I·E- strains, delete TCR VPI I bearing cells in the thymus (6 3).

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ZAMVTL & STEINMAN


EAE-A PROTOTYPE FOR T CELL-MEDIATED AUTOIMMUNE DISEASE Tremendous strides toward a better understanding of autoimmune patho genesis have been made from studies of EAE. This model of antigen induced autoimmunity has been successful for several reasons: (a) Of the models for autoimmunity, EAE has a long history, having been developed earlier than other models. (b) EAE is easily induced in many species. (c) Myelin basic protein (MBP), the primary autoantigen in EAE, abundant in eNS tissue, is easy to extract and purify. In contrast with other auto antigens such as acetylcholine receptor (experimental autoimmune myas thenia gravis) or collagen (collagen induced arthritis), M BP is exceedingly small; murine M BP is approximately 1 7 kd. This small size of MBP has facilitated the identification of critical determinants. (d) The clinical mani festations of EAE, namely paralysis, a similarity to human multiple scler osis, and loss of tail tone (rodents), are readily apparent to most observers. Other additional steps, such as the measurement of antibody levels as in experimental autoimmune myasthenia gravis or in experimental autoimmune thyroiditis, are not necessary for clinical assessment in this model.

A Historical Perspective of EAE The discovery of EAE can be traced back to the turn of the century, when there was concern regarding the adverse "allergic" responses to the original rabies vaccine. Individuals given the Pasteur treatment (developed in 1 885), which consisted of fixed rabies virus grown in rabbit eNS tissue, approxi mately 0 . 1 % of recipients developed acute disseminated encephalomyelitis (ADE), a monophasic paralytic illness (64). Histopathologic examination demonstrated perivascular infiltrates of mononuclear cells and focal areas of demyelination within the central nervous system (eNS). Since ADE was not a sequelae of rabies infection, and individuals not exposed to rabies but given the Pasteur treatment also developed these "paralytic accidents," it was hypothesized by Remlinger (65) in 1 905, that eNS tissue con taminating the vaccine preparation precipitated ADE. Stuart (64) later showed that rabbits immunized with either normal rabbit or human spinal cord tissue developed ADE (acute EAE), with the same clinical and histo logic features observed in individuals experiencing postvaccinal paralytic attacks. Thus, eNS tissue, and not fixed virus alone" was considered the agent responsible for precipitating postvaccinal encephalomyelitis. In classic studies Rivers (66, 67) showed a demyelinating encephalomyelitis was induced in monkeys by repeated (> 50) injections of normal rabbit eNS tissue (67). When Freund's adjuvant was used to emulsify eNS tissue,



587

fewer ( < 3) injections were necessary for induction of EAE in monkeys (68). In 1 949 Olitsky (69) was able, using adjuvants, to induce demy elinating EAE in mice, thus establishing murine EAE as a model for CNS demyelinating diseases, such as multiple sclerosis. Since then acute EAE has been induced in several species including mice, rats, guinea pigs, monkeys, sheep, dogs, and chickens (70). Kabat in 1 947 (71) suggested that EAE may have an autoimmune etiology. He suggested that the autoantigen(s) were located in the white matter, since injection of fetal CNS tissue, lacking myelin, did not cause EAE. Myelin basic protein (MBP), the major protein constituent of myelin (approximately 30% total protein), was identified as an encephalitogenic antigen in CNS tissue by Einstein in 1 962 (72). The amino acid sequence of MBP from various species has been determined (73). MBP has been shown to induce acute EAE in mice, rats, guinea pigs, rabbits, and monkeys, and in humans accidentally injected in the course of laboratory experiments (70). Encephalitogenic activity of various MBP fragments has been examined by several investigators using different EAE models. MBP nonapeptide 1 1 4- 1 22 causes EAE in guinea pigs (74). M BP 68-88 is ence phalitogenic in rats (75-77). Initial studies in mice demonstrated that M BP 1 -37 and C-terminal 89- 1 69 causes EAE in separate inbred mouse strains (4 1 , 78, 79).

The Lymphocyte in EAE In 1 960 Paterson demonstrated that EAE could be induced in naive recipi ent rats by adoptive transfer of lymphocytes (80). "Anti-lymphocyte" antisera was shown to inhibit induction of EAE by Waksman in 1 96 1 (8 1 ). Amason et al (82) demonstrated that neonatal thymectomy prevents EAE, suggesting a critical role for thymus derived lymphocytes. This was clearly established by Gonatas & Howard in 1 974 (2) and Ortiz-Ortiz & Weigle in 1 976 (1) who independently demonstrated that thymocytes were required for induction of EAE in recipients depleted of T cells by thy mectomy and irradiation. With the availability of T-cell subset markers it became clear that TH lymphocytes mediated EAE. Petinelli & McFarlin in 1 98 1 (83) demonstrated that elimination of M BP-primed Ly 1 + T cells, but not elimination of Lyt 2+ (cytotoxic-suppressor subset) cells, pre vented transfer of EAE to naive recipients. Ben-Nun and others demon strated that continuous lines of M BP-reactive TH cells could adoptively transfer EAE (84-88). Further evidence for the critical role for TH cells in EAE included the observation that TH cells are found in abundance in inflammatory EAE lesions in the CNS (89) and that monoclonal antibodies to CD4, the marker expressed by class II-restricted T H cells, can inhibit or reverse ongoing EAE in rats (90) and mice (91).

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T-Cell Clones Mediate Autoimmune Encephalomyelitis The techniques for generating antigen-specific T-cell clones were developed in 1 980 by Kimoto & Fathman (92). By utilizing this technique to generate T-cell clones specific for the autoantigen M BP, it was hoped one could elucidate pathogenic mechanism(s) in EAE. Encephalitogenic clones pro vide several advantages over heterogeneous M BP reactive T cells. One can: (a) examine the specific activation requirements and mechanism(s) for autoimmune destruction by a single MBP-reactive T cell; (b) examine lymphocyte homing to the CNS, the site of pathogenesis; (c) identify and characterize encephalitogenic MBP epitopes recognized by individual T cells; (d) clarify class II-restricting elements and class-II associations with susceptibility to EAE; (e) examine TCR gene usage of individual ence phalitogenic T cells. With this knowledge one could envision the develop ment of more specific forms of immune intervention in EAE. At the time we began our studies with M BP-reactive T-cell clones, Ben Nun and colleagues had demonstrated that continuous MBP-reactive T cell lines, but not clones isolated from those lines, could adoptively transfer acute EAE to naive recipient rats (84-87). Our studies were initially based on an observation made by Fritz and colleagues (78). They had demon strated that the N-terminal 1 -37 amino acid (aa) peptic fragment of rat or guinea pig MBP could induce EAE in H_2u mice, PL/J and B I O.PL, while the C-termina1 89-1 69 aa fragment was encephalitogenic in H-2s strains, SJL/J and A.SW (4 1 , 78). T-cell clones were isolated from PL/J and (PLSJ)F 1 mice immunized with native rat or bovine M BP. All clones isolated were of the T H subtype, being CD4 + and class-II restricted. These clones were characterized for their class II-restriction patterns and reac tivity to M BP fragments. The possible class II-restricting elements in these strains are shown in Figure 1 . Different patterns of M BP reactivity were observed (Table 1), confirming that there are multiple T-cell determinants of M BP. T-cell clones restricted to Aa uAfJu and Aa 'AW recognized the encephalitogenic guinea pig MBP 1 -37; however, only those MBP 1 -37specific T-cell clones restricted to I -A U(Aa U AfJU) recognized a determinant shared with mouse (self) M BP. Guinea pig and rat M BP contain His1 0 and Gly 1 1 (73) which is deleted i n mouse M BP (Figure 2). We predicted that clones recognizing an epitope shared with mouse (self) MBP would be encephalitogenic, but clones responding to determinants of rat or guinea pig M BP, but not mouse MBP, would be unlikely to induce EAE in mice. When recipient (PLSJ)F I mice were injected with N-terminal, 1 -37-specific, T-cell clones restricted by I-A", EAE was observed in 1 00% of mice injected (93, 94). The onset and severity of EAE was dependent upon the number of T cells injected. When greater numbers of cloned T

EXPERIMENTAL ALLERGIC EN CEPHALOMYELITIS

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H_2u H-2S Possible hybrid class II molecules in H_2(U x s) F, mice

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Possible class n molecules expressed in PL/J, SJL/J and (PLSJ)F I mice. Parental PL/J express I-A "(Arx"A{3") and I-E"(Erx"E{3U) class II molecules. Parental SJL/J mice express I-N(Arx'A{3') only; Erx is deleted in H-2' strains ( 1 58). (PLSJ)F I can express parental class II

Figure I

molecules and may express hybrid class II molecules, Arx"A{3', Arx'A{3u, and Erx"E{3'.

cells were injected, the onset was earlier and the course of EAE was more severe . With injection of 5 x 106 encephalitogenic cloned T cells, EAE developed in approximately· 1 I days; the mean day of onset with 1 06 cells was 15 days; with 5 x 1 05 cells the mean onset was 24 days; and with 1 05 cells the mean onset was 44 days. Mice injected with 5 x 1 06 T cells developed acute fulminant EAE which resulted in death. However, when injected with smaller numbers of cells, a majority (70%) of recipient mice developed relapsing paralysis, a feature not observed in rat EAE. Approximately 30% of recipient mice developed chronic unremitting para plegia, a form not usually described. The clinical course of representative individual recipient mice is shown in Figure 3 . I n other studies o f murine EAE, i t had been demonstrated that low dose

10

20

30

Rat/Guinea Pig MBP

Ac-A 5 Q K R P 5 Q R H G 5 K Y L A T A 5 T iii! 0 H A R H G F L P R H ROT G I

Mouse MBP

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Bovine MBP

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N terminal amino acid sequences of MBP from various species. Reprinted with permission from the Journal of Experimental Medicine (94).

590

ZAMVIL & STEINMAN

Patterns of antigen specificity and class-II restriction of (PLSJ)F I M BP-specific T-cell clones


Table 1

Pattern

Class-II restriction

II III IV

I-A(AauAfJU) I-A(Aa'A{3') I-A(Aa'A{JU) 1-E(EauE{J')

Recognition of MBP \-37

Recognition of mouse MBP

+

+

+ +

Reprinted with permission from the Journal of Experimental Medicine (94).

whole body irradiation and administration of Bordetella pertussis vaccine facilitated induction of T cell-mediated EAE (95). We have found that low dose irradiation (350 rad), but not B. pertussis, facilitates induction of EAE with MBP-reactive T-cell clones (93). However, injection of greater quantities of cloned encephalitogenic T cells obviates the need for X irradiation. All mice injected with 1 07 developed chronic EAE (Table 2). One could also avoid the requirement for irradiation with fewer cloned T cells by in vivo administration of recombinant IL-2 for 4 days (S. Zamvil, L. Steinman, unpublished observations). Recipient mice have been examined for histologic features of EAE. In mice who developed EAE, examination at the light and electron micro scopic levels revealed classic histologic evidence of encephalomyelitis including perivascular infiltration by mononuclear cells within the white matter but not the gray matter of the eNS. Meningeal inflammation, demyelination, and axonal degeneration were also observed. Remy elination by oligodendrocytes was also seen (93, 94). These results demon strated for the first time that clone.s which respond to a defined self-antigen could induce clinical and histologic autoimmune disease. It has since been demonstrated that T-cell clones which recognize other determinants of mouse (self) MBP can cause clinical and histologic EAE in mice (39, 96).

Encephalitogenic and Non-Encephalitogenic T-Cell Clones R ecognize MBP: Mechanism of Pathogenesis? Two classes of nonencephalitogenic M BP-specific T-cell clones were identi fied. T-cell clones that recognized determinants of rat MBP (the original immunogen), which were not shared with mouse (self) MBP, did not cause clinical or histologic signs of EAE on adoptive transfer to mice, even when injected with more than 1 07 cells. This class of T cells included those T cell clones that recognize the encephalitogenic N-terminal 1 -37 fragment of rat and guinea pig MBP, but not mouse M BP (Table 1 , pattern III).


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DAYS AFTER INJECTION OF PJR-25

Figure 3 Clinical observation of individual (PLSJ)F I recipient mice following injection of T-cell clone PJR-25. Representative mice from each of the following groups are shown: A (105 cells); B & C (5 X 1 0'); D & E ( 1 06); F (5 X 106). Clinical disease was graded as follows: 0, no observable signs of clinical disease; 1 , loss of tail tone only; 2, mild paraparesis; 3, moderately severe paraparesis; 4, complete paraplegia; 5, moribund; t, death. In vivo administration of encephalitogenic T cell clones has been described (93, 94). Reprinted with permission from the Journal ojExperimental Medicine (94).

The second class of non-encephalitogenic clones expressed the same class II-(I-N)-restriction as encephalitogenic clones and recognized M BP 1 -37, including mouse (self) M BP (93, 94). These results established that T-cell recognition of mouse M BP was necessary but not sufficient for induction of EAE in mice. The difference in encephalitogenic potential was not a function of Ag/MHC recognition because non-encephalitogenic


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p,o Table 2

on

Induction o f EAE with an enceph alitogenic M BP-specific T cell clone Irradi ation"

Cell number I

5 I

5 I a

x

X

x

x x

107 1 06 106 1 05 1 05

Sick/total mice" 1 0/ 1 0 1 0/ 1 0 20/20 1 0/ 1 0 1 0/ 1 0

No irradiationb

Onset (days)

Mean seve rity

Number sick/total mice

Onset (days)

Mean severity

9

5.0 5.0 4.2 3.0 2.5

1 0/ 1 0 4/10 1/10 OjlO NT

30 31 45

4.2 4.0 2.5

II

15 24 44

(PLS1)F 1 mice were injected with encephalitogenic T-cell clone P1R-25 a s described previously (93, 94). Recipient mice received 350 rad whole body irradiation

4 hr prior to administration o f P1R-25 cells.

h(PLSJ)F1 mice were injected with encephalitogenic Tcell clone P1R-25. These recipient mice did not receive whole body irradiation.

� �



593

T-cell clones could not be distinguished from encephalitogenic T-cell clones on the basis of fine MBP-specificity (97). Furthermore, these clones utilized the same TCR genes as determined by Southern analysis of TCR f3 chains (57) and sequence analysis of TCR IY. and fJ chain genes (58, 98). Other properties of MBP-reactive T cells, in addition to antigen recog nition, must contribute to their encephalitogenic potential. Clones that cause relapsing paralysis and demyelination may have a greater capability to migrate specifically or "home" to the CNS. When examined on a fluorescence-activated cell sorter (FACS) for expression of MEL- 1 4 (a cell surface marker associated with lymphocyte homing to high endothelial venules-99), none of the encephalitogenic or non-encephalitogenic clones expressed MEL- 1 4 (94). These clones do not differ in TH subtype (97, 1 00); all encephalitogenic and non-encephalitogenic clones are THl , secreting gamma-interferon and IL-2, but not IL-4. It is not known whether injected M BP-reactive T cells directly mediate the pathogenic damage within the CNS. Encephalitogenic T cells may interact with other cells, including macrophages, B-cells, and plasma cells, which have been observed within EAE lesions ( 1 0 1 ) . Some investigators have proposed that the encephalitogenic T cells may initiate demyelination ( 1 02). One group of investigators have demonstrated that astrocytes, induced to express class II in vitro, can present MBP to MBP-specific T cell lines ( 103), and M BP-specific rat T-cell lines can mediate lysis of astrocytes in vitro ( 1 04). Thus far we have been unable to demonstrate cell-mediated lysis in vitro with activated or resting encephalitogenic T cell clones. However, our most recent studies suggest that production of certain cytokines may be relevant to the pathogenesis of T cell clone induced EAE. Although both encephalitogenic and nonencephalitogenic T-cell clones secrete gamma interferon and IL-2, in a select group of clones there was a positive correlation between encephalitogenic potential and expression of the TNFfJ (lymphotoxin) gene and production of TNFfJ protein (Powell, Zamvil, Lederman, Ruddle Steinm an, submitted for publication). Further studies are clearly necessary to elucidate the mechanism(s) of autoimmune demyelination and the role of individual cytokines. ,

T-Cell Epitopes and Class-II Restriction in EAE The isolation of MBP reactive T-cell clones which mediate EAE has facilitated the identification of individual encephalitogenic epitopes. The encephalitogenic T-cell epitope within MBP 1 -37 was the first to be identi fied ( 1 05) and has been characterized in greatest detail. We had initially observed that separate forms of native (intact) MBP, varying in their N terminal sequences, differed in their ability to stimulate individual M BP


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ZAMVIL & STEINMAN

1 -37 specific, I-N-restricted encephalitogenic clones to proliferate in vitro. In fact, bovine M BP, which was known to be less encephalitogenic in PL/J mice (93), was found to be less stimulatory than rat or mouse M BP. Since bovine M BP 1 -37 differs from the mouse M BP 1 -37 sequence only at residues 2 and 1 7 (see Figure 2), it was predicted that the epitope recognized by these clones would include one of these two residues. Using overlapping synthetic peptides containing these two residues we identified the epitope to be located within the first 1 1 residues. Peptides p l - 1 1 and p l - 1 6 were equipotent with intact rat or mouse MBP. Shorter peptides, p l -7 and p l -9, were less stimulatory ( 1 05). A few features of this T-cell epitope were intriguing. First, acetylation of residue 1 , NAc-Ala, was essential for recognition for all N-terminal MBP-specific encephalitogenic clones. Furthermore, the amino acid Ala I was necessary, since peptide p2- 1 1 did not stimulate these clones. Second, peptides containing Ala2 instead of Ser2 were less stimulatory to all clones tested regardless of which N-terminal peptide or form of intact MBP was used as the original immunogen. This substitution, which is present in bovine MBP, may explain why bovine MBP is less encephalitogenic in PL/J and (PLSJ)F) mice. Third, all of the N-terminal M BP reactive T-cell clones expressed the same phenotypic specificity, responding to each of the N-terminal peptides in the same manner. This observation was extended by Mitchell & Wraith, who tested synthetic peptides containing single alanine substitutions at each position from residue 1 to 9 (48). All of the clones tested responded to these additional substituted pep tides in a similar manner. A second epitope located within the N-terminus was identified. As described earlier, nonencephalitogenic T-cell clones isolated from homo zygous PL/J or (PLSJ)F) mice, restricted by AauA[3u or Aa uA[3" respec tively, recognize MBP 1 -37 of rat or guinea pig M BP, but not mouse (self) M BP. Rat and guinea pig MBP contain His 1 0 and Gly ll, which is deleted in the mouse MBP sequence (see Figure 2). The epitope recognized by these clones is located within residues 9- 1 6 ( l 05). Within N-terminaI 1 - 1 6 residues o f M B P there are two distinct M BP T-cell epitopes. Certain N-terminal M BP peptides were observed to cause acute EAE. Immunization with M BP peptides containing residues 1 -9, 1 - 1 1 , and 1 - 1 6, all of which are recognized by encephalitogenic N-terminal-specific clones, induced EAE. In contrast, peptides that did not stimulate encephalitogenic clones did not induce EAE in H_2u or H_2(uXS) F) mice. Thus, there was a strict concordance between recognition in vitro and potential to induce EAE, suggesting that the repertoire of encephalitogenic N-terminal M BP specific T cells is limited to a discrete population. Furthermore, as we observed for encephalitogenic T-cell clones, all p I - I I primed T cells from



595

H_2u and H_2(uxs) F1 mice were restricted by the same class-II molecule, I-Au (97). In an analogous manner, the encephalitogenic region located within 891 69, the fragment which causes EAE in H-2S strains, was identified. This epitope was predicted using Rothbard's template for peptide recog nition (36). The encephalitogenic epitope, p89- 1 0 1 ( 1 06), contains the tetramer 90-93(HFFK). In contrast with the encephalitogenic response to the N-terminus, there is more than one discrete population of ence phalitogenic I-A" restricted T cells for SJL/J mice. Two distinct, although overlapping, encephalitogenic peptides were identified by our group, p891 0 1 and p89- 1 00 ( 1 07). One group of encephalitogenic clones recognizes both p89- 1 0 1 and p89- 1 00, while another group requires the presence of residue 1 0 1 for proliferation, responding to p89-101 but not p89-100 ( 1 07). Both peptides are encephalitogenic in SJL/J mice. A distinct ence phalitogenic peptide for SJL/J mice, p96- 1 09, was identified by Kono et al ( l08) (see Table 2). Another I-A" restricted encephalitogenic T-cell epitope of MBP may exist. An SJL/J M BP specific T-cell line that recog nizes p 1 7-27 causes EAE in recipient syngeneic mice ( l 09). Direct immun ization with p 1 7-27 will confirm whether p 1 7-27 is indeed an encepha litogenic T-cell epitope. Based on an observation that certain T-cell clones recognize epitopes of mouse (self) M BP distinct from MBP 1 -37 and 89- 1 69, we predicted that cryptic encephalitogenic epitopes existed. An epitope recognized by an encephalitogenic T-cell clone, restricted to a hybrid I-E molecule (Table I, pattern IV), was identified. This T-cell epitope, p35-47, also contains a sequence, 42-45 (RFFS), predicted from Rothbard's template (36). Peptide p35-47 causes EAE in H_2U mice PL/J and (PLSJ)F I mice with the same severity observed with MBP p l - l l in these strains. T-cell recognition of this epitope is restricted by homozygous I-E (EiXUEpU) and hybrid I-E (EiXuE{JS) (see Table 2). These results demonstrated for the first time that antigen-specific T cells restricted by I-E molecules, the homolog of human HLA-DR (17), can mediate autoimmune disease (39). More recently an I-E-restricted encephalitogenic T-cell epitope of MBP has been identified in the rat model of EAE ( 1 1 0). The identification of these encephalitogenic and nonencephalitogenic epitopes emphasizes the complexity in studying EAE. It is now clear that there are multiple discrete encephalitogenic epitopes of MBP. T-cell recognition of each occurs in association with a separate allelic class II restriction element. Another myelin antigen, proteolipid protein (PLP), also causes acute EAE (11 1 ). A T-cell line directed against PLP can adoptively transfer EAE. An encephalitogenic region, p 1 39- 1 5 1 , has been identified for SJL/J mice. Susceptibility to several human autoimmune

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diseases, including insulin-dependent diabetes mellitus, pemphigus vul garis, and multiple sclerosis, is linked to more than one class-II gene (25). Separate autoantigens or multiple discrete T-cell epitopes of a single autoantigen, as our studies have demonstrated, could, in part, account for the association of more than one class-II (HLA-D) gene with susceptibility to certain autoimmune conditions (39).


Differential Susceptible to EAE A large body of scientific research has been devoted to the identification of polymorphic differences in class-II structure and their relationship to susceptibility to autoimmunity (26). Increased class-II expression is also thought to be critical in the pathogenesis of autoimmune disease, especially if class II is expressed aberrantly where it is not constitutively present ( 1 1 2). The relative contribution of individual class-II expression in indi viduals that are heterozygous for class-II loci has not been extensively investigated. Fritz and colleagues (79) made the intriguing observation that while the MBP fragment 1 -37 was encephalitogenic in PLjJ, and MBP fragment 891 69 caused EAE in SJLjJ mice, the encephalitogenic response in (PLSJ)F 1 mice was not codominant; MBP 1 -37, but not MBP 89-[69, caused EAE in (PLSJ)F 1 mice. Two possibilities were proposed at that time to account for this finding. First, the lack of in vivo response to MBP 89- 1 69 could be due to a defect at the T-cell level. In other words, I-A'-restricted T cells recognizing MBP 89- 1 69 could be rendered unresponsive, by suppression or inactivation. The second possibility, not mutually exclusive with the first hypothesis, was that the level of!-A' expression by (PLSJ) F I antigen presenting cells was insufficient for effective priming to MBP-reactive I-A'-restricted T cells. The latter possibility seemed much more likely. Differential expression of combinatorial I-E molecules in hybrid mice had been observed. In F I mice, when one parent was H_2U (either PLjJ or B IO.PL) and the other parent was from another haplotype, there was a preferential cis association of Ea" with Ef3", to create relatively more parental EauEf3 u parental heterodimers ( 1 1 3). This differential expression of I-E molecules influenced the in vitro response of cytochrome c-reactive T cells (1 1 4). Differential expression of I-E molecules was demonstrated in other F, hybrids by examination of cloned alloreactive T cells ( 1 1 5) and antigen-specific T cells ( 1 1 6). Both possibilities have been investigated by our group and Fritz and his coworkers (94, 1 1 7, 1 1 8). We had generated MBP specific T-cell lines and clones from (PLSJ)F 1 mice immunized with rat M BP. A clear bias in class II restriction emerged; with both the initial MBP-primed T-cell lines and -



597

the clones from these lines, we observed a paucity of proliferative T cells recognizing MBP in association with I-A' class-II molecules (94). The deficiency in I-A' restriction was not specific for M BP ( 1 1 7). Fritz demon strated that the relative deficiency of I-A' restricted T cells occurred in (PLSJ)F, mice immunized with ovalbumin, purified protein derivative (PPD), or Listeria monocytogenes, antigens which induce T cell-pro liferative responses in homozygous SJL/J mice. These results were in support of the hypothesis that there was limited expression of I-A' in (PLSJ)F, mice. Two lines of evidence gave further support. First, (PLSJ)F, responder T cells were tested for their ability to give a mixed lymphocyte response to parental APC. Since T cells are tolerant of self-MHC antigens, the reasoning was that if I-A" expression was not deficient in (PLSJ)F, mice, (PLSJ)F, T cells would not respond to I-A'-bearing APC in a classic mixed lymphocyte response. Whereas culture of (PLSJ) F , responder T cells with PL/J stimulator cells did not generate a significant response, culture with SJL/J stimulator cells caused a strong MLR ( 1 1 8). An obser vation of similar significance was made for H_2(uxq)F, mice. Whereas H_2(uxq)F, responder T cells did not proliferate to the H_2U stimulator cells, they did proliferate to H-2q stimulator cells. The second, and most convincing, line of evidence that there was a lack of I-A' expression in (PLSJ)F, mice came from the direct examination of I-A' expression by (PLSJ)F, APC. Using a monoclonal antibody that recognizes a private specificity ofI-A', Fritz & Skeen found no significant I-A" expression on the surface of (PLSJ)F, antigen presenting cells ( 1 1 8). Furthermore, by two-dimensional gel electrophoresis, all four a and f3 chains were present. This raises the possibility that a majority of I-Aas or I-Af3 is expressed as hybri d H_2UXS F, molecules. Consistent with this possibility is our observation that most T-cell clones isolated from (PLSJ) F, mice are restricted to AasAf3u class-II molecules (94; unpublished observations). Observations of deficient I-A expression in F, mice were made in another model. Wicker & Hildeman ( 1 1 9) examined the response to Trinitropbenyl mouse serum albumin in BIO ([H-2b] [responder]), B I O.A ([H-2a], [1N][nonresponder]), and (B I 0 x B l O.A)FI mice. In H_2bxk F, mice, non responsiveness was dominant. By examination with allele-specific mono clonal antibodies, Sandrin et al ( 1 20) observed a relative lack of expression of I-Ab in H_2bxk F, mice. It is interesting that although deficient I-A" expression in (PLSJ)F, mice has been demonstrated, functional I-A" expression by (PLSJ)F , antigen presenting cells has been seen. We (94) and others ( 1 1 8) have shown that I-A" restricted SJL/J or (PLSJ)F 1 T-cell clones respond to all forms of MBP when cultured in vitro with (PLSJ)F, APC. A possible explanation


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for this finding is that these APC have become activated by gamma inter feron, or another cytokine released by MBP-specific T cells, to express increased amounts of I-N. In the study of H_2bxk F I splenic APC, Sandrin et al found that although expression of l-Ab was deficient in the resting state, activated splenic APC expressed I-Ab ( 1 20). Another finding, indi cating that sufficient I-A' can be expressed in vivo, is that p89- 1 0 1 , which causes EAE in SJL/J mice ( 1 06), also induces EAE in (PLSJ)F I mice (S. S. Zamvil, unpublishcd observation). Furthermore, McCarron & McFarlin ( 1 2 1 ) have shown that an encephalitogenic MBP 89- 1 69 specific (PLSJ)F I T-cell line could be generated by expansion on SJL/J APC, and this I-A' restricted T-cell line was encephalitogenic in (PLSJ)F I mice. Differential expression of class II has been observed in another model of EAE. Although strain 2 guinea pigs are resistant, strain 1 3 and strain (2 X l 3) F I guinea pigs are susceptible to EAE ( 1 22). The vascular endo thelial cells within strain (2 x 1 3) F I guinea pigs express quantitatively more strain 1 3-specific than strain 2-specific class II molecules. However, normal tissue within strain (2 x 1 3)FI guinea pigs did not show this difference. Whether this phenomenon is due to different activation require ments for gene expression or is explicable at the protein level is not clear. Using a genetic approach, Germain and colleagues ( 1 23) have inves tigated why "free association" of alpha and beta chains does not always create equal quantities of I-A heterodimers. They examined the expression of I-A in L-cells transfected with various I-Aa and I-Af3 chain genes. In one example, using a recombinant beta chain gene, they mapped control of I-A expression to the f31 domain. Their results suggested that individual I-AaAf3 chain pairing was influenced by polymorphic residues within the first domain of I-A chains. It is possible that preferential I-A pairing may be relevant to these examples in which deficient allelic I-A expression is found in F I mice. The results from studies involving hybrid mice in EAE demonstrate a further level of complexity in predicting immune responses based on MHC genotype. The inability to respond to MBP 89- 1 69 was not predictable from the genotype. The basis for this observation appears to be a relative deficiency of expression of allelic I-A' in H_2UXS F I mice. In human auto immune disease susceptibility may sometimes be recessive in individuals heterozygous at class-II loci. In insulin dependent d.iabetes mellitus (IDDM), an autoimmune d.isease associated with HLA-DR3 and -DR4, heterozygous individuals expressing HLA-DR2 and H LA-DR3 or -DR4 are less susceptible. In this example, DR2 has been referred to as "pro tective" for IDDM (26). In IDDM and other diseases, it has yet to be determined whether susceptibility in individuals heterozygous at class II


599

is explicable on the basis of quantitative differences in expression of specific allelic and hybrid class-II molecules.


T CELL RECEPTOR GENE EXPRESSION IN EAE The identification of multiple encephalitogenic epitopes of MBP indicated that the potential repertoire of M BP-specific T ceIls includes more than one population of T cells. However, the T-cell response to each epitope appears limited to discrete populations of T ceIls. For example, ence phalitogenic N-terminal MBP-specific T-ceIl clones could not be dis tinguished from one another on the basis of their reactivity to peptides of MBP or class-II restriction. Furthermore, there was a concordance between in vitro T-cell recognition and encephalitogenic potential after active immunization. Both of these results suggested that the TCR reper toire of encephalitogenic N-terminal M BP-specific T cells in H-2u was limited. Recent advances in molecular biology have made it possible to examine the T cell receptor of individual T cells. With this technology it is possible to examine TCR gene expression of T cells mediating EAE, and to address whether T cells that appear phenotypically similar in their Ag/MHC recognition express common TCR genes. TCR gene expression has been examined for the encephalitogenic response to MBP 1 -9 and MBP 89- 1 0 1 .

The Encephalitogenic N- Terminus TCR gene expression of M BP p 1 -9 specific T ceIls has been examined by three approaches: (a) cell surface staining with TCR Vp-specific mono clonal antibodies; (b) Southern analysis; and (c) TCR gene sequencing. T cell clones from PL/J mice were initially stained with monoclonal anti bodies specific for TCR Vp8 (62, 1 24, 1 25). TCR Vp8 is a three-member family ofTeR genes encoding TCR (62, 1 24) expressed by several strains, including PL/J. This TCR gene family is deleted in certain strains, including SJL/J and SWjR. When a panel of 1 8 p l -9 specific T-cell clones, isolated from 1 4 separate PLjJ mice, were stained with these antibodies, 1 4 (78 %) expressed TCR Vp8 (57). This high percentage was a minimum estimate, as potential "sister" clones from a single T-cell line were excluded in this calculation. When all clones were included, 85 % expressed TCR Vp8 ( 1 26). In many strains, including PL/J and B I O.PL, Vp8 is the predominant TCR Vp family expressed, accounting for 1 6-25 % of peripheral T cells. Therefore, we asked whether the high frequency of usage of TCR Vp8 was an in vitro cloning artifact, or whether it represented VP8 usage in vivo. Secondly, we examined whether the use of TCR Vp8 was specific for M BP


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1 - 1 1 . Lymph node cells from MBP 1 - 1 1 primed PLjJ mice were sorted by FACS into CD4+ jVPS+ and Cd4+ jVPS- subpopulations. When stimulated in vitro, > 90% of the proliferative response to MBP I - I I occurred in the VpS + subpopulation. Thus, the high fn�quency of VpS usage was not a cloning artifact. Furthermore, I-EU--restricted ence phalitogenic T-cell clones specific for p35-47 are VP8 - (57). When p3547 specific CD4+ jVP8 + and CD4+ jVP8 - subpopulations were exam ined in primary cultures, the proliferative response occurred within the TCR V{38 - subpopulation (58). However, TCR V{38 usagl� in the response to MBP is not specific for M BP 1 -9. When independent I-A"-restricted T-cell clones specific for the non-encephalitogenic epitope, p5- 1 6, were examined with monoclonal antibodies specific for TCR V {38, six of seven (84%) utilized TCR Vf38 (39) . These results are also intriguing in that earlier studies by Morel (6 1 ) demonstrated that most V{38 + T-cell clones specific for myoglobin were I_Ed restricted in DBAj2 (H_2d) mice. From their results, they suggested that V{38 usage correlates with I-E restriction. If Vp usage does correlate with class-II restriction, based on our results, Vf38 expression may correlate with I-A restriction in PLjJ mice. Heterogeneity in the T-cell response to M BP 1 -9 was further evaluated by molecular genetic techniques. By Southern analysis, V{38.2 was identi fied as the TCR V{3 gene used by VpS + T cell clones (57, 58). This was confirmed by the sequencing of TCR genes of eight MBP 1 -9 specific T cell clones. Of these eight clones, seven utilized Vf38.2; one encephalitogenic clone expressed V{34 (Table 3). There was less restrictive use of D{3 and J{3, with four clones utilizing J{32.7, two using J{32.3, and two clones expressing JP2.5. Thus, the predominant V{3-J{3, expressed by 4 (50%) of Table 3

Peptide p I- I I

Multiple discrete T cell epitopes of myelin basic protein Encephalitogenic

Class-II

potential

restriction

+

Aa"Af3u

References

Va

Vf3

Va4.2

Vf38.2

57, 58, 105, 1 27

Va2_3 p5- 1 6

AauAf3u, Aa'Af3u

ND

Vf38+

39, 97

p 1 7-27

?

Aa'Af3'

ND

ND

109

p35-47

+

EauEf3u, EauEf3'

ND

Vf38-

39

p89- 1 00

+ + +

Aa'Af3'

ND

ND

1 06, 1 07

Aa'Af3'

ND

Vf3 1 7

1 08, 109

Aa'Af3'

ND

ND

p89- 1 0 1 p96- 1 09



60 1

these clones, was V(J8.2-l(J2.7. Even less heterogeneity was observed in rx chain gene usage. AU eight clones used the same Vrx, Vrx4.3, new member of the Vrx4 family (also referred to as VrxP1R-25, Acha-Orbea 1 989). Six of these clones utilized lrxTA3 1 , one used lrxTT l l , and one used lrxF I - 1 2. The predominant V rx-lrx, expressed by six (75%) clones was Vrx4.3-lrxTA3 1 (Table 4). Thus, there was a striking degree of restriction in the rx and (J chain TCR gene usage in response to the encephalitogenic N-terminus. TCR gene expression for MBP 1 -9 specific T cells was examined in another H-2u strain, B I 0.PL ( 1 27, 1 28). This strain contains the same MHC, the H-2U haplotype, on a B I 0 background. As in PL/J mice, MBP 1 -9 is encephalitogenic in B 1 O.PL and p 1 -9 specific T cells are restricted by I_Au ( 1 05). Of 33 MBP 1 -9 specific hybridomas, 79% utilized V(J8.2 with 1{32.7 (referred to as J{32.6 or 1{32.7, depending upon whether or not the sixth J gene of the J{32 cluster, a pseudogene, is considered in the numerical order) (53, 58), and 21 % used V{3 1 3 with J{32.2. Although {3 chain gene usage was very similar to that seen for PL/l p l -9 specific T cell clones, rx chain gene expression was somewhat different. In contrast with the PL/l clones analyzed, all having used Vrx4.3, of the B l O.PL clones examined, 58% used Vrx2.3 and 42% expressed Vrx4.2. Both Vrx2.3 and Vrx4.2 bearing T-cell hybridomas utilized the same 1 gene, lrx39 (58, 1 27). Within PL/l and B l O.PL mice, the expression of TCR genes in the MBP p l -9 specific response is quite strikingly limited. However, when comparing TCR gene expression between these two strains, certain differences were apparent. Even though V{38.2 is used to the same extent by both strains, it is Table 4

Summary of TCR sequences

Clone

Vf3a

Jf3

Va

Ja

PJB-20 PJpR-2.2 PJpR-6.2 F J 21

8.2 8.2 8.2 8.2

2.7 2.7 2.7 2.7

PJR-25 PJR-25 PJR-25 PJR-25

TA3 1 TA3 1 TA3 1 TA3 1

PJR-25 PJB - 1 8

8.2 8.2

2.3 23

PJR-25 PJR-25

TA3 1 TA3 1

Group 2

.

PJpR-7.5

8.2

2.5

PJR-25

TTI I

Group 3

FJ-12

4

2.5

PJR-25

FJ-12

Group 4

-

Group I

a The nomenclature system for TCR elements by Wilson et al (51) was used. VaPJR-25 is a new member of the Va4 family and has 93% ,nucleotide sequence homology to Va TA65 (58;98). JaF 1 - 1 2 has 95% homology in nucleotide sequence to Ja5CC7 (53). The clones were divided into groups on the basis of V{JJ{J combinations. Reprinted with permission from Cell (58).

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unclear why Va2.3, which was not expressed by any of the PL/J clones, was used more frequently than Va4 in B l O.PL mice. Polymorphic differences in TCR gene expression may exist between these strains. Clones expressing different Va genes may differ in their p l -9/I-N affinity. If so, they may differ in their proliferative capability and/or in vivo function. Furthermore, it is not clear which individual B l O.PL T cells, used to derive T-cell hybridomas, were encephalitogenic. Although we have found no difference in TCR gene usage by encephalitogenic and nonencephalitogenic p 1 -9-specific T-cell clones in PL/J mice, this has not been established for T cells from B I0.PL mice.

The Encephalitogenic C- Terminus TCR gene usage in the encephalitogenic T-cell response of SJL/J mice to the C-terminus has been examined, although not as extensively as for M BP p l -9. The T-cell response appears more complex. Three 'encephalitogenic peptides have been identified, p89- 1 0 1 , p89- 1 00, and p96- 1 09 ( 1 06-1 08). TCR V{3 expression has been examined for T cells that respond to p891 0 1 . Approximately 50% of T cells which proliferate to p89- 1 0 1 also respond to p89- 1 00. The other 50% require Pro l O l for stimulation. TCR V{3 gene expression for these two populations has been examined with a monoclonal antibody that recognizes V{3 1 7, a single gene family, expressed by several I-A + /I-E- strains, including SJL/J ( 1 9, 20). Interestingly, all clones that recognize p89- 1 0 1 , but not p89- 1 00, use TCR V{3 1 7. All clones that proliferate to p89- 1 00 are V{3 1 7 - ( 1 07). The TCR VfJ(s) expressed by V{3 1 7 - clones is not known at this time. Examination of TCR a chain genes and further analysis of the {3 chain genes is currently in progress. Examination of susceptibility to EAE in different strains indicates that MHC genotype, and not TCR repertoire, control susceptibility induced with MBP pS9- 10 l . H-2' (I-A') strains, SJL/J and A.SW, and H-2q (I-Aq) strains, SW/R and B l O.T(6R) strains that differ in non-MHC genes, are all susceptible to EAE induced with M BP p89- l O I . Sequence analysis of Aa ( 1 29) and AfJ ( 1 30) suggest that I_AS and I-Aq are very similar. Interest ingly, SJL/J and SW/R have deleted approximately 50% of their V{3 genes, including V{38. However, these two strains express V{3 1 7. In contrast, A.SW and B I O.T(6R) express V{38 but not V{3 1 7. Thus, susceptibility in this case does not correlate with the absence of V{38 or the expression of V{31 7. By examination of transgenic mice expressing various "susceptible" class-II genes, it may be possible to assess the relative contribution of MHC and TCR repertoire in individual encephalitogenic responses.

EAE in the Rat The Lewis strain is the most extensively studied rat in EAE. In this strain, MBP p68-88 contains an encephalitogenic determinant, although another



603

encephalitogenic epitope(s) probably exists ( 1 3 1). Encephalitogenic T cells specific for p68-88 are CD4 + and class II-restricted, although there is some ambiguity in the identification of the exact class-II restricting element ( 1 32). Approximately 50% of CD4 + T-cell clones raised against rat MBP are specific for p68-88 ( 1 3 1). The TCR composition of rat T cells specific for p68-88 has been examined by TCR sequencing of one clone and by subsequent probing of other clones by Northern and Southern analysis. The TCR data from the rat clearly supports previous studies of MBP p 1 9 specific T cells i n H-2u mice, demonstrating a marked restriction i n usage of Vrx and V{3 in the T-cell response to an individual encephalitogenic determinant ( 1 33). Seventy percent of p68-88 specific T-cell hybridomas utilize the same Vrx, and 1 00% expres� the same V{3 gene. Interestingly, the Vrx is 77% homologous to Vrx2, one of two Vrx's utilized by MBP p l 9 specific mouse T-cell clones. The V{3 used b y these clones i s most closely related to mouse Vf38.2, sharing 80% homology. Although there is con siderable homology between the Vrx and Vf3 genes used in rat and mouse T cell clones, rat T cells do not recognize MBP p l -9 on H_2u APC, and conversely, mouse T cells do not respond to p68-88 cultured with rat APC ( 1 32). Offner et al ( 1 1 0) described an I-E restricted sequence p87-99 of rat MBP that is encephalitogenic in the Lewis rat. The TcR recognizing p87-99 in the context of I-E also appears to express Vrx2 and Vf38. Because of the similarity in V rx and V f3 gene usage between Lewis rats and H_2u mice, Heber-Katz ( 1 32) has suggested that TCR V-region usage is the "defining entity" in EAE, independent of Ag/MHC. Although this hypothesis is intriguing, it does not account for all encephalitogenic T-cell epitopes. In mice multiple distinct encephalitogenic T-cell epitopes include p l -9 , p35-47, and p89- I O l . Thus, within one species, it is known that at least three separate Vf3 genes are used in encephalitogenic T-cell responses to MBP (Table 3). Nevertheless, the TCR data from the rat clearly supports previous studies of recognition of M BP p l -9 in H_2u mice in at least one aspect: there is a marked restriction in usage of Vrx and Vf3 in the T-cell response to an individual encephalitogenic determinant.

IMMUNOTHERAPY OF EAE Prevention and Treatment of EAE With A nti- TCR Monoclonal Antibodies The monoclonal antibody (MAb) F23. 1 depletes Vf38-positive T cells from the peripheral blood ( 124). T cells reactive with MAb F23 . 1 constitute 25% of the T cells in lymph nodes of normal PL/J mice. In the (PLSJ)F I mouse this percentage is 14 % . The depletion of T cells reactive with MAb


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F23 . l is 98% complete 3 days after intraperitoneal (ip) administration of a dose of 0.5 mg (58). EAE was first induced EAE with T-cel1 clone P1R-25. This clone is ful1y encephalitogenic, capable of inducing paralysis and demyelination (93, 94). P1R-25 expresses the epitope recognized by MAb F23 . 1 (57). Therapy was begun 24 hr after the mice first developed paralysis. In two experiments (Figure 4) (PLS1) F 1 mice were randomly divided into two groups, with 1 6 mice each receiving two 1 00 Ilg injections of F23 . 1 i.p. at 72 hr intervals, while 1 6 mice received MAb Leu 5b (S5.2), an isotype-matched control reactive with the CD2 antigen (a pan T-cel1 marker on human but not on mouse T cells). Within 2-4 days mice receiving F23 . 1 showed a marked reversal in their paralysis, and 1 3 out of 16 were completely free of disease 10 days after therapy started. Only one relapse with tail weakness was seen, on day 35, in the animals given MAb F23 . l . Next we tested whether EAE induced with p l - l l i n CFA in (PLS1)F , mice could be prevented with MAb F23. 1 . Immunization with MBP pep tide p I - I I in CFA can induce clones which are both F23 . 1 -positive and -negative, and which are fully encephalitogenic. Successful prevention of disease with F23 . 1 would indicate that the F23 . 1 -positive T cell clones predominate in the development of disease and that the depletion of these T-cel1 clones in vivo would not simply result in an escape to F23 I -negative T-cell clones that would cause disease. Results shown in Table 5 indicate that whereas 1 of 19 mice receiving MAb F23 . I developed EAE, 9 out of .

�

a: w > w t/)

Days after injection of F23.1 Figure 4 Reversal of T cell clone induced EAE with mAb 523 . 1 . (PLSJ)F I mice were injected intravenously with 5 x 106 cells of encephalitogenic T cell done PIR-25. Mice became paralyzed within 2-3 weeks and were randomized for treatment with mAb F23. 1 on mAb S5.2 (anti-Leu 5b). Two 1 00 JIg i.p. injections of either F23 . l or S5.2, respectively, were given at 24 hr and 96 hr after paralysis was first observed. Severity: 0, no signs of clinical disease; I, limp tail; 2, paraparesis; 3, paraplegia. Filled boxes represent S5.2 treatment, open boxes F23. 1 treatment. Reprinted with permission from Cell (58).

60S


Table 5

Prevention of MBP-peptide p i- I I induced EAE with MAb F23. 1 . Reprinted with permission from Cell (58) Monoclonal antibody"

Incidenceb

Clinical disease mean onset (day)

1/19 9/20

20 1 5c

F23. 1 S5.2

' MAbs F23.1 or SS.2 were given i.p. (SOO pg) on days - I , I, and 9, where immunization with p l - l l was on day O. b The ratio of number of paralyzed mice to the total number of mice. All mice were examined through day 40.


C

The standard deviation was 1 .7 days.

20 mice given MAb SS.2 became paralyzed (p < 0.001). These results serve to indicate that the V f38-expressing clones predominate functionally in the induction of EAE. (PLSJ)F I mice were immunized with guinea pig MBP. In (PLSJ)F I mice there are at least two distinct encephalitogenic epitopes for MBP, p l - l l and p3S-47. The response to p3S-47 is restricted to I-E" and involves mostly Vf38-negative T cells. After paralysis was present, mice were given 0.2 mg i.p. of the MAb F23 . 1 or KJ23a, a monoclonal antibody specific for the product of the TCR Vf3 1 7a gene product ( 1 9, 20). KJ23a prevents EAE induced with T-cell lines responsive to MBP p89- 1 0 1 in the SJL mouse (S. Zamvil, unpublished results). Of 1 9 (PLSJ)F 1 mice given F23 . 1 , 1 2 returned to normal within 72 hr, while 2 1 of 2 2 mice given KJ23a had moderate to severe paraplegia after 72 hr (Table 6). Relapses were seen in S of 19 F23 . 1 -treated mice in the next 14 days. Thus treatment with F23 . l reversed EAE i n a situation where mUltiple encephalitogenic epitopes were Table 6

Reversal of guinea pig MBP induced EAE with mAb F23. 1 . Reprinted with permission from Cell (58) Number ofmice with clinical symptoms 72 hr after treatment" Treatment" F23. 1 KJ23a

Number of mice with clinical symptoms 1 4 days after treatment

None

Mild

Severe

None

Mild

Severe

Deaths

12 1

5 12

2 9

14 9

3 2

7

4

' Treatment was begun 24 hr after mice exhibited EAE. At this time the mice were separated randomly into two groups. Mice in each group received one 200 pg i.p. injection of F23 . 1 or SS.2. Nineteen mice received F23.1 and 22 mice received KJ23a. b Clinical status was graded as follows: none, no neurologic symptoms; mild, flaccid tail and/or mild paraparesis; severe, severe paraparesis or complete paraplegia.

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present. Vf18-negative T cells capable of responding to MBP p I - I I or p3547 may have accounted for the relapses seen in the F23 . 1 -,treated mice. In contrast, the SJl (I-A') mouse strain recognizes a peptide from M BP (p89- 1 0 1 ) with at least three overlapping epitopes. There is evidence for limited TCR gene usage in recognition of one of these epitopes ( 1 08, 1 09). However, depletion of this subset ofT cells did not prevent antigen-induced EAE; elimination of a single Vf1 subset, in a polyclonal autoimmune disease such as this, may not be sufficient to prevent or reverse disease.


Prevention and Treatment of EAE by T-Cell Vaccination Cohen and associates have shown that it is possible to use autoimmune T-cell clones or lines as vaccines to prevent or reverse autoimmune disease. An inoculum of T-cell clones below the threshold for triggering disease, or irradiated, fixed, or pressure-treated T cells can serve as vaccines (85, 1 34-1 36). These animals remain free of disease for prolonged times, and EAE could not be induced with T-cell lines, T-cell clones, or with M BP in adjuvant. T-cell clones specific for EAE-inducing T-cells have been isolated from rats that recovered from EAE, suggesting an anti-·idiotypic mech anism for protection. These T-cell clones are either CD4+ or CD8 + . The CD8 + T cells lyse their targets specifically, and this cytotoxicity is not blockable with anti-CD4, anti-CD8, anti-class I or anti-class II antibodies. Recently, EAE in the Lewis rat was prevented by immunization with a nonapeptide spanning the V-D-J region of V p8, expressed on about three fourths of T-cell clones recognizing encephalitogenic M BP p72-86 ( 1 37). Vandenbark and associates protected against EAE with a peptide from the CDR2 region of Vp8 ( 1 3 7a).

Prevention and Treatment of EAE With Anti-Class II Antibodies In 1 98 1 it was demonstrated that EAE could be prevented by injection of anti-I-A prior to immunization with spinal cord homogenate ( 1 38). Anti I-A treatment reduced the influx of radio labeled lymphocytes that home to the CNS in EAE ( 1 39). When anti-I-A treatment was given after the first appearance of paralysis in EAE, mice return to normal within 48 hr. Anti-I-A treatment also reduced the number of relapses and mortality in chronic relapsing EAE ( 1 40). In rhesus monkeys treatment of paralytic disease was successful with polymorphic mouse anti-HLA-DQ or HLA DR antibodies that react with Rh-LAD ( 1 4 1 ). Therapy with monoclonal anti-class II antibodies is partially specific, only blocking responses restric ted by a given class-II isotype. Thus, although anti-I-A blocks EAE, EAMG, and thyroiditis, in each of these diseases responses to PPD were left intact ( 142, 1 43).


607


Prevention and Treatment of EAE With Anti-CD4 Antibodies Reversal of EAE with noncytotoxic anti-CD4 antibodies was demon strated by Brostoff & Mason (89) in rats, while cytotoxic anti-CD4 anti bodies successfully reversed EAE in mice (90). Construction of chimeric mouse-rat anti-CD4 antibodies has demonstrated the enhanced efficacy of cytotoxic constructs. Chimeric antibodies derived from the variable region of GKl . 5, a cytotoxic rat antimouse CD4 MAb, with various mouse Ig constant region isotypes have been designed. While GKl . 5 and its chimeric derivatives GK I .5y l , GK I .5y2a, GK I . 5y2b were cytotoxic, GK I .5y3 was not. In treatment and reversal of EAE, GK1 .5y3 was distinctly less effi cacious than GK1 . 5, GK 1 . 5y l , or GK 1 . 5y2a ( 1 44).

Prevention of EAE With Peptides Blocking TCR-MHC Interaction Recent experiments have shown how the induction of an immune response can be "blocked" with nonimmunogenic peptides ( 145). The feasibility of blocking autoimmune disease by peptide competition for TCR-MHC i nteractions has also been established. We showed that the nonstimulating nonacetylated 1 -20, or Ac9-20, can inhibit the induction of EAE caused by acetylated p I - I I . Coinjection of competitor peptides prevented para lytic EAE at competitor to inducer ratios of 3 : 1 to 6 : 1 ( 1 46). The likely mechanism underlying this observation is that the competitor peptides inhibit the binding of the encephalitogenic peptide Ac 1 - 1 1 to I-Au. More potent competitors have been designed that also block EAE. Putative contact sites for TCR and for I -AU have been identified and a nonimmunogenic peptide that binds with a thousand-fold higher affinity to I-N was used to prevent EAE ( 1 47). Seia and Arnon and associates have treated EAE with a random copoly mer termed CopI of tyrosine, alanine, lysine, and glutamate. This peptide was successfully employed in therapy of relapsing-remitting MS ( 1 48). Recent experiments suggest that CopI blocks MHC binding of myelin ( 149) .

MULTIPLE SCLEROSIS AND EAE EAE is often considered our best model for the human disease multiple sclerosis (MS) (3, 1 50). Although similarities exist, there are several distinct differences (Table 7). MS is a demyelinating disease of the eNS, which affects approximately 250,000 people in the United States. Although characterized by relapsing-remitting paralysis, the clinical course is quite

608


Table 7

ZAMVIL & STEINMAN

Similarities and differences between EAE and MS

Relapsing and chronic paralysis CNS demyelination Linkage to MHC class I I CD t T cells i n CNS perivascular inflammatory lesions T-cell mediated pathogenesis Autoantigen(s) Restricted TCR V-gene usage" Spontaneous disease

EAE

MS

+ + + + + MBP, PLP

+ + + + ? ? ? +

+

' Epitope specific restricted TCR V-gene usage as described in text.

variable, some individuals developing acute fulminant MS, and others developing chronic progressive MS. The histologic hallmark of MS is the presence of focal areas of demyelination within the white, but not the gray, matter. MS is a disease of young adults with onset commonly between 1 5 and 40 years of age, with a female to male ratio of 1 .5 : 1 ( 1 5 1 ). Several lines of evidence support both a genetic predisposition and an immune etiology. Although not mutually exclusive, epidemiologic evidence also suggests that MS involves an infectious agent, acquired before the age of 1 5 ( 1 5 1).

Genetics of Multiple Sclerosis The evidence supporting a genetic predisposition is compelling. First, second, and third degree relatives of MS patients are at increased risk of developing MS, accounting for the familial aggregation of MS known to exist. MS twin studies demonstrate a much greater concordance in monozygotic, as opposed to dizygotic, twins ( 1 52). The risk of developing MS for a monozygotic twin of an MS patient is at least 25%, compared to a 2% risk in dizygotic twins, or nontwin siblings of MS patients. The results of one study examining the Ig allotypes in MS patients suggest that certain immunoglobulin gene allotypes may influence susceptibility to MS ( 1 53). As for susceptibility to EAE, susceptibility to MS is associated with certain MHC alleles. MS is more common in HLA-DR2 individuals, although there is overrepresentation of HLA-B7, -CW l , and -DQWI in MS patients (5, 1 54). The association between MHC ancl susceptibility to disease indirectly suggests that T cells are involved in disease pathogenesis. By RFLP analysis of TCR rx chain genes, polymorphic fragments associ ated with both MS and myasthenia gravis have been identified ( 1 5 5). A recent study examining the inheritance of TCR f3 chain genes of 40 sibling pairs concordant for relapsing-remitting MS demonstrated a significantly


609

increased representation of a particular TCR f3 haplotype compared to unaffected sibling pairs ( 1 56). These studies suggest that TCR f3 chain genes or other genes in linkage disequilibrium with the TCR f3 gene complex contribute to susceptibility to MS. Beall et al described an association between susceptibility to MS and a TcR V{3 gene in patients who are HLA DR2Dw2 ( 1 57).


T Cells in Multiple Sclerosis Histologic evidence also suggests that T cells participate in MS. As in EAE, the central nervous system (CNS) lesion of chronic MS is characterized by perivascular infiltrates of lymphocytes and macrophages associated with demyelination ( 1 50). Large numbers of CD4+ and CD8+ T cells are found at the lesion margin, while CD4 + , but not CD8 + , T cells are found in adjacent normal white matter ( 1 58). Further analysis with monoclonal antibodies which distinguish T-helper-inducer (THI) from T-suppressor inducer (TSI) indicate that the CD4 + T cells in active demyelinating lesions are of the TH I subset ( 1 59). Several immune abnormalities have been observed in the peripheral blood and cerebrospinal fluid (CSF) of MS patients. In the analysis of peripheral blood from chronic progressive MS patients, it has been reported that there is a loss of TSI (CD45R + ) T cells ( 1 60) . This finding however. was not specific for MS, as patients with systemic lupus erythem atosus also have reduced numbers of CD45R + T cells in peripheral blood. Peripheral blood lymphocytes of MS patients have been examined for the presence of TA I , a putative marker for memory T cells ( 1 6 1 , 1 62). These cells were also found to be increased in peripheral blood. Analysis of CSF lymphocytes in MS patients has revealed an overrepresentation of CD4 + T cells. Although not to the same extent, increased CD4+ T cells were found in the CSF of patients with other neurologic diseases ( 1 62) . More recent studies of CSF lymphocytes using monoclonal antibodies for T81 (CD45R +) and T HI (CDw29 + ) subsets, ' indicate an accumulation of CD4jCDw29+ T cells with a paucity of CD4jCD45R + T cells ( 1 63, 1 64). It has been suggested that the synthesis of oligoclonal IgG, one of the hallmarks of MS, may be indirectly related to decreased TSl function. Another experimental approach to study the role of T cells in MS has been the direct eX'amination of the TCR of CSF T cells in MS patients ( 1 65, 1 66). In one stu'dy CD4+ T cells were isolated from the CSF of progressive MS patients, cloned in vitro, and examined by Southern analy sis with TCR J{3 and tf3 probes. Common TCR gene rearraQgements were found among CSF T cells, but not peripheral blood ,T .cells, suggesting that an oligoclonal population of T cells may be involved in the inflam matory eNS process ( 1 66). Interestingly, by a similar approach, oligo'

610

ZAMVIL & STEINMAN

clonal T-cell proliferation has been found in the synovial membranes of rheumatoid arthritis patients, another nonmalignant inflammatory disease ( 1 67). Unfortunately the Ag/MHC specificity of the T cells in these pro cesses is not known. Although the possibility of oligoc1onality is provoca tive, these results have not provided substantial insight into the identi fication of the target autoantigen(s) in these inflammatory processes.


Myelin Basic Protein in Multiple Sclerosis Several investigators have examined the T cell response to MBP and other possible myelin autoantigens ( 1 60, 1 68- 1 74). T cells specific for MBP have been identified in the peripheral blood of patients with MS and acute disseminated encephalomyelitis, and from the CSF of MS patients ( 1 68). Although to a lesser extent, MBP reactive T cells were also observed in controls and patients with other neurologic diseases. Furthermore, T cells can be cloned from the blood and CSF of MS patients, as well as the peripheral blood of normal individuals ( 1 60, 1 69, 1 7 1 , 1 75). Using pep tides of M BP, investigators are currently examining whether MS patients may respond to different epitopes than do controls, and whether these M BP reactive T cells from MS patients share common Ag/MHC specificity ( 1 72- 1 7 5). The response to other myelin antigens in MS patients has also been examined. In one study examining peripheral blood lymphocytes, MS patients responded to myelin-associated glycoprotein as well as M BP, but reactivity to proteolipid protein was not detected in MS patients ( 1 70). Although reactivity to myelin antigens can be detected in MS, their sig nificance remains unknown. Even if we accept that these T cells are involved in MS, what is their role? Unlike in EAE, where we can demon strate that MBP reactive T-cell clones can initiate autoimmune demy elination, we do not know if these myelin reactive T cells in MS are causative, or if they occur secondarily.

An Infectious Etiology in Multiple Sclerosis Epidemiologic studies have suggested an infectious, possibly viral, etiology in MS. Although reports are conflicting, at one time or another several viruses including vaccinia, mumps, rubella, herpes simplex I ( 1 5 1 ), and HTLV I ( 1 76, 1 77) have been implicated in the pathogenesis of MS. Infection with Theiler's virus and the JHM strain of mouse hepatitis virus causes encephalomyelitis and demyelination in mice, and visna virus causes demyelination in sheep ( 1 5 1 , 1 78). It is unclear to what extent demy elination is due to direct viral infection or to the immunologic response to the virus.



61 1

It is difficult to assimilate the genetic, epidemiologic, and immunologic observations into one coherent model. Although several attempts have been unsuccessful in identifying an infectious agent, this failure does not preclude the possibility that a virus or another infectious agent contributes to the pathogenesis of MS. Of the several possible explanations which have been proposed ( 1 5 1), two are appealing. First an infectious agent, possibly with tropism for the CNS may cause damage to oligodendrocytes or another cellular constituent, or possibly damage the blood brain barrier. CNS cellular damage or loss of integrity of this organ barrier, may allow release of CNS "auto" antigens, not normally exposed, in an immunogenic form. This exposure, secondary to infection, may lead to a self-per petuating inflammatory process with expansion of self-antigen specific T cells causing increased class-II expression, facilitating further presentation of autoantigen(s). The virus, or another infectious agent, while necessary for initiating this cascade, may not remain present or remain latent as a provirus. Because of the transient nature of infection, viral products may not be detected and evidence of past infection is based on serology or techniques demonstrating latent proviral genes. Another model for spontaneous autoimmune disease, which has received widespread interest in the past few years, is based on the immuno logic cross-reactivity between pathogens and self proteins. In this model of "molecular mimicry," infection may lead to immunologic responses directed against determinants of a pathogen that share structural similarity or amino acid sequence homology with self antigens, inducing an "anti host" (autoimmune) process. In this regard decapeptides of measle, influ enza, adenovirus, and EBV that share homology with human MBP have been identified ( 1 79). Fujanami & Oldstone ( 1 80) have found evidence supporting molecular mimicry in EAE. Using an eight amino acid peptide of hepatitis B virus polymerase sharing six residues (75 %) in common with encephalitogenic site of MBP for rabbits, they showed that rabbits immunized with this viral peptide developed histologic, but not clinical signs of EAE. Another example of molecular mimicry in T cell-mediated autoimmune disease has been found in the model, experimental auto immune uveitis ( 1 8 1). Although theoretical, molecular mimicry is very attractive as it may account for viral, genetic, and immunologic con tributions in autoimmune pathogenesis. Studies ofmolecular mimicry have been limited in the past by insufficient knowledge of viral protein sequences and lack of identification of encephalitogenic epitopes of MBP. As multiple encephalitogenic epitopes of M BP have been recently identified, and pro tein sequences of viruses known to cause demyelination are becoming available, molecular mimicry will be explored thoroughly in murine models of EAE.

612

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CONCLUSION EAE is a prototype for T cell-mediated autoimmune disease. Of the several well-characterized models of antigen-induced T cell-mediated auto immune diseases, including experimental autoimmune myasthenia gravis, experimental autoimmune thyroiditis, collagen-induced arthritis, adju vant-induced arthritis, and experimental autoimmune uveitis; EAE has been explored the most thoroughly. Examination of the T-cell response to the small autoantigen myelin basic protein has revealed that there are-. multiple encephalitogenic epitopes, whoserecognition is restricted by sep arate allelic class-II (immune response) molecules. As for T-cell recognition ' of foreign antigens, which involves a ternary interaction of nominal antigen with MHC and TCR, studies in this model for autoimmunity have estab lished a direct functional relationship between MHC, TCR, and the epitopes of MBP. It thus appears the rules that govern T-cell recognition of foreign antigen also apply to the effector T cell recognition of an autoantigen. EAE has served as a model for testing novel forms of immunotherapy. Antibodies that block formation of the ternary complex, such as MAb directed at appropriate class II or TCR, as well as other antibodies to nonpolymorphic T-cell surface markers have been shown to prevent or treat EAE. More recently it has been demonstrated that peptide analogues of encephalitogenic determinants, which probably compete for class-II binding, can prevent EAE ( 1 47, 1 83, 1 84). Furthermore, one group has demonstrated that by tolerizing a host to an encephalitogenic determinant, one could prevent subsequent EAE in an epitope specific manner ( 1 85). Each of these new therapeutic modalities was based on recently acquired knowledge of the immunogenetic constituents in EAE, emphasizing the importance of examining and further defining the cellular and genetic components involved in autoimmune pathogenesis. It is hoped that by identifying the T cells involved in MS and other diseases, defining their ' recognition of MHC and self antigen, the same forms of therapy, effective in EAE, may be tested in human autoimmune disease. Literature Cited

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42.

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Role of CD8+ T cells in murine experimental allergic encephalomyelitis.

Chronic experimental allergic encephalomyelitis.

Hyperphenylalaninaemia and experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis in aged F344 rats.

Cerebrospinal fluid lymphocytes in experimental allergic encephalomyelitis.

Reactive and nonreactive lymphocytes in experimental allergic encephalomyelitis. I. Possible role of macrophage-lymphocyte interactions.

Experimental autoimmune uveoretinitis (EAU) versus experimental allergic encephalomyelitis (EAE): a comparison of T cell-mediated mechanisms.

Alkyllysophospholipid prevents induction of experimental allergic encephalomyelitis.

Multiple Sclerosis and Experimental Allergic Encephalomyelitis.

Sympathectomy augments adoptively transferred experimental allergic encephalomyelitis.

Modulation of experimental allergic encephalomyelitis by somatostatin.

Apoptosis in the nervous system in experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis in cynomolgus monkeys. Quantitation of T cell responses in peripheral blood.

Shared T-cell receptor gene usage in experimental allergic neuritis and encephalomyelitis.

Cinnamon ameliorates experimental allergic encephalomyelitis in mice via regulatory T cells: implications for multiple sclerosis therapy.

In trans T cell tolerance exacerbates experimental allergic encephalomyelitis by interfering with protective antibody responses.

T cell sensitization to proteolipid protein in myelin basic protein-induced relapsing experimental allergic encephalomyelitis.

t-PA acts as a cytokine to regulate lymphocyte-endothelium adhesion in experimental autoimmune encephalomyelitis.

Role of antibodies to galactocerebroside in experimental allergic encephalomyelitis.

Progressive demyelination and reparative phenomena in chronic experimental allergic encephalomyelitis.

Prevention and treatment of murine experimental allergic encephalomyelitis with T cell receptor V beta-specific antibodies.

Myelin basic protein treatment of experimental allergic encephalomyelitis in monkeys.

Peripheral nervous system pathology in relapsing experimental allergic encephalomyelitis.

Reactive and non-reactive lymphocytes in experimental allergic encephalomyelitis.