Intervention with immunomodulatory agents: T cell vaccination

12 Intervention with immunomodulatory agents: T cell vaccination G A B R I E L L E KINGSLEY G A B R I E L S. P A N A Y I

A E T I O P A T H O G E N E S I S OF R H E U M A T O I D ARTHRITIS

The current treatment of rheumatoid arthritis (RA) is generally agreed to be unsatisfactory since the drugs used are both toxic and frequently ineffective in preventing disease progression. In the light of the many recent advances in our understanding of the immunopathogenesis of RA, it should now be possible to move from empirically based to rationally developed therapies by targeting cells and molecules known to be involved in the disease process. Such developments are particularly relevant to patients in the early phases of the disease since the initiating immune-inflammatory processes, rather than secondary phenomena, are likely still to be operative. Although our understanding of R A is incomplete, our knowledge is sufficient to develop a model (Figure 1), permitting delineation of potential therapeutic targets (Kingsley et al, 1991). The experimental support for this T cell-centred model has been reviewed in detail elsewhere (Kingsley et al, 1990; Panayi et al, 1992). Suffice it to say here that several lines of evidence can be adduced to support a pivotal role for T cells in the pathogenesis of RA. First, immunopathological examination of the synovium in RA demonstrates an infiltrate composed of mainly CD4+ T cells clustered around antigen-presenting cells (APCs), whereas normal synovium contains no T cells (Janossy et al, 1981). Second, in reactive arthritis, an inflammatory arthritis in many respects similar to RA but where the causative antigen is known, T cells specific for the triggering agent can be found in the joint (Sieper et al, 1991). Third, in animal models of arthritis, such as adjuvant arthritis (AA), the disease can be transferred from diseased to naive animals by T cells. Fourth, the main function of major histocompatibility complex (MHC) class II antigens is to present antigenic peptides to T cells both in the periphery during an immune response and in the thymus during repertoire selection; thus the association of R A with H L A DR4 and DR1 (Lanchbury, 1988; Wordsworth et al, 1989) also argues for the importance of CD4+ T cells in RA. The final and most convincing evidence comes from the experience with early T cell-directed immunotherapies like thoracic duct drainage (Paulus et al, 1977), lymphocytapheresis (Emery et al, 1986) and Bailli~re's ClinicaIRheumatology--

Vol. 6, No. 2, June 1992 ISBN0-7020-1636-5

435 Copyright9 1992,byBailli~reTindall All rights ofreproductionin anyformreserved

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Antigenpresentation T Migration 'Activated' Regulatory j~l~k CD4*i cells mechanisms 'Elfector'mechanisms~ ~es'egridw~t ,bhOof i~dr~Su'n~eed'1lasers ~ omt ~ ~nflaJm~

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total lymphoid irradiation (Gaston et al, 1983) which, although impractical on a large scale, were effective. In the model of RA (Figure 1), an unknown rheumatoid antigen (exogenous or autoantigenic) is presented, probably by a restricted range of RA-associated MHC class II antigens (DR1 and subtypes of DR4), to CD4+ 'arthritogenic' T cells, which become activated. In rodent autoimmune diseases such as experimental allergic encephalomyelitis (EAE), the disease-inducing T cells have been found to be clonal, or at least to use a limited number of T cell receptor (TCR) V[~ regions (Acha-Orbea et al, 1988). However, although a restricted T cell repertoire might be anticipated in RA, not only in the light of the animal evidence but also on theoretical grounds, no definite conclusion has yet been reached (Paliard et al, 1991; Uematsu et al, 1991; Bowness and Bell, 1992). Following activation, the arthritogenic CD4 + T cells trigger an immuno-inflammatory cascade, which involves a variety of effector ceils (T cells, APCs, B cells, synoviocytes) and mediators (cytokines, growth factors, enzymes), leading to synovial inflammation, proliferation and joint destruction. It is still not clear whether RA can be initiated by more than one antigen or whether the original

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antigen or another, for example a cross-reacting autoantigen, is responsible for the subsequent perpetuation of the disease. It is also uncertain whether the initial T cell activation occurs in the joint, systemically or at both sites, but in any case T cells will need to migrate into the joint, providing another possible therapeutic target. An autoimmune response also triggers a variety of regulatory events aimed at re-establishing normal self-tolerance. As yet these are not fully defined but have great potential relevance since enhancement of natural specific immunoregulatory mechanisms could provide a long-lasting innocuous therapy (Kroemer and Martinez-A, 1991). POSSIBLE TARGETS FOR IMMUNOTHERAPY

How does this information help in the design of new immunologically based therapies for RA? Current treatments, such as disease-modifying agents, glucocorticoids and cytotoxic drugs, are directed, not at the apex of the pyramid, the interaction between rheumatoid antigen, disease-causing T cell and disease-associated MHC molecule, but at less specific mechanisms at lower levels. Furthermore, these drugs are purely immunosuppressive, yet amelioration of R A might be achieved more permanently and less deleteriously by stimulating the normal regulatory mechanisms which control autoaggression rather than solely by suppression of proinflammatory events (Kroemer and Martinez-A, 1991). Recently developed experimental therapies for RA (Kingsley et ai, 1991; Kroemer and Martinez-A, 1991) such as cyclosporin, cytokine antagonists, immunotoxin-interleukin 2 conjugates and monoclonal antibodies against the T cell surface molecules, CD4, CD5 and the interleukin 2 receptor are somewhat more selective in their immunosuppressive effects and thus potentially less toxic. However, even inhibitors which act directly on the disease-associated MHC allele, such as allele-specific monoclonal antibodies or peptides displacing the autoantigen from the MHC (Wraith et al, 1989), are still selective rather than truly specific since any MHC allele presents more than one antigen. Most importantly, these immunosuppressive treatments all share the disadvantage that relapse is likely on withdrawal; so therapy must be prolonged. In contrast, treatments which induce tolerance, either by depletion, functional inactivation or active suppression of autoaggressive T cells, could lead to long-lasting remission. However, to avoid permanent immune deficiency, they should ideally be specific. If the arthritogenic T cells in R A proved to be oligoclonal, they could, as in EAE, be deleted using anti-TCR monoclonal antibodies (Acha-Orbea et al, 1988). Antigen-specific T cell anergy can be induced by giving antigen in a non-immunogenic way, for example orally (Thompson and Staines, 1990), but this cannot be done in RA because the antigen is unknown. A final way to restore self-tolerance would be to establish cognate immunosuppression by generating suppressor cells (anti-idiotypic suppressor T cells) which recognize the T cells implicated in the autoimmune pathology. As we shall see, this is precisely what happens as a result of T cell vaccination.

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WHAT IS T CELL VACCINATION? T cell vaccination (TCV) is based on the same principle as immunization used to prevent infectious diseases, where protection is established by vaccination with a non-pathogenic form of the causative organism or its products. The vaccine is rendered non-pathogenic either by development of an attenuated form of the microbe which retains sufficient immunological similarity to be protective (for example vaccinia virus immunization against smallpox) or by chemical treatment of the microbial product (for example the use of tetanus and diphtheria toxoids). In both cases, the immunized subject will develop protective immunity without becoming ill. In a similar way vaccination with T cells can be applied in autoimmune diseases which are T cell mediated. In TCV (Figure 2), the disease-inducing (autoimmune) T cells, in an activated and attenuated form, are injected into the subject, who develops a specific T cell immune response against them. If he subsequently begins to develop the disease, this immune response will suppress the disease-inducing T cells and thus protect him from disease. Furthermore, TCV is also effective in down-regulating an ongoing autoimmune response; it can thus also be used as treatment, which is of paramount importance in human autoimmune disease. The concept of TCV was initially evolved by Cohen and colleagues (Cohen et al, 1985; Cohen, 1986; Cohen and Weiner, 1988) in animal models of autoimmune disease such as EAE and AA following the observation that animals which had recovered from these conditions were

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relatively resistant to a second attack. These studies will be considered in the initial part of this chapter. The second section will examine the evidence that vaccination with TCR peptides alone, rather than whole T cells, can provide protection. Finally, the application of both techniques to human autoimmune disease will be explored.

TCV IN ANIMALS Protection against disease in autoimmune animal models

The first studies of TCV were performed in a roden~ model for multiple sclerosis (MS), EAE, which is a paralytic disease induced by injection with myelin basic protein (MBP). EAE is T cell-mediated since it can be transferred to naive recipients by T cells from primed animals, and is usually self-limiting. Ben-Nun et al (Ben-Nun et al, 1981a; Ben-Nun and Cohen, 1982) isolated an MBP-specific T cell line, Zla, from Lewis rats with EAE which was able to cause EAE when injected into naive rats. They subsequently showed that Z l a cells, attenuated by irradiation or treatment with mitomycin C, could protect rats against the development of EAE (Ben-Nun and Cohen, 1981; Ben-Nun et al, 1981b), provided that the Z l a cells were activated (Naparstek et al, 1983) either by MBP itself or non-specifically with concanavalin A. Thus, Z l a in this form acted as a TCV. TCV studies were also performed in AA, another T cell-mediated animal model with many features similar to RA. AA is induced in rats by injection of Mycobacterium tuberculosis (MTb) in complete Freund's adjuvant (CFA); the causative MTb protein is now known to be the MTb 65 kDa heat shock protein (HSP 65) (van Eden et al, 1988). In initial experiments (Holoshitz et al, 1982), an MTb-specific T cell line, A2, was derived from rats with AA. This line could mediate AA in naive recipients, albeit only if their sensitivity to A A had been enhanced by total body irradiation. Attenuated forms of A2 were shown to protect against AA whether the disease was induced by T cell transfer or by MTb/CFA. The protection could be transferred from vaccinated to non-vaccinated rats by transfer of thymus or spleen cells (Holoshitz et al, 1985). Since A2-treated rats were resistant to AA but not EAE, the protection was not due to generalized immunosuppression. Because protection was specific for A2, and did not occur with various control T cell lines, the authors proposed that it resulted from immunization against the TCR (the molecule which distinguishes between T cells with differing antigenic specificities). In further work Cohen's group have also found TCV to be effective in other models of induced autoimmune disease, notably autoimmune thyroiditis (Maron et al, 1983), and in a spontaneous animal autoimmune disease, diabetes in the non-obese diabetic (NOD) mouse (Cohen, 1991a). Other groups have confirmed the efficacy of TCV. For example, in collagen-induced arthritis (CIA), Kakimoto et al (1988) isolated a T cell line capable of propagating and, after attenuation by irradiation, vaccinating against CIA.

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Development of a protective T cell clone To enable more detailed delineating of the mechanisms involved in mediating TCV-induced protection, the A2 cell line was cloned and an arthritogenic T cell clone, A2b, was derived (Holoshitz et al, 1984). A2b proliferates not only to a nine amino acid peptide from positions 180-188 of the MTb 65 kDa HSP (van Eden et al, 1988) but also to an autoantigen, cartilage proteoglycan (van Eden et al, 1985). This cross-reactivity has been proposed to explain the pathogenesis of A A , but the exact connections between A2b, MTb HSP 65, cartilage proteoglycan and AA remain unclear (Cohen, 1991a). For example, immunization of animals with the 65 kDa HSP suppresses A A in an antigen-specific manner but is not arthritogenic (Billingham et al, 1990); furthermore, animals preimmunized with the 180-188 peptide are prevented from developing AA, probably via a T cell-mediated specific immunosuppression (Yang et al, 1992). Like its parent line, A2b protected against AA when administered in a nonpathogenic form (Lider et al, 1987); protection was disease specific since induction of EAE was normal. T cell vaccination in the treatment of established disease Using the A2b system, further important advances from the standpoint of human autoimmune disease were made. A2b was shown not only to protect against A A but also to treat established disease. In these experiments (Lider et al, 1987) rats were immunized with MTb and, 15 days later when all were suffering from AA, were inoculated either with A2b or the control encephalitogenic line Zla. Rats injected with Z l a developed A A of similar duration and severity to unvaccinated animals; by contrast, in rats vaccinated with A2b the disease was virtually aborted. In practical terms, this means that TCV can be used not just to protect against disease but also to treat ongoing conditions, which is much more relevant to the human situation. T cell vaccination using lymph node cells as vaccines In human autoimmune diseases, where the causative antigens are not clearly defined, the pathogenic T cells are unknown. Thus the demonstration (Lider et al, 1987) that suitably activated and attenuated unseparated lymph node (LN) cells from MTb-primed animals can act as effective vaccines was very important. Vaccination with primed LN cells was shown to be immunologically specific since LN from animals with A A protected against A A but not E A E and vice versa (Lider et al, 1987). Furthermore, Mor et al (1990) were able to enrich the proportion of antigen-specific cells in such LN populations without using specific antigen by culturing with concanavalin A followed by interleukin 2. The mechanism by which concanavalin A, normally considered a pan-T cell mitogen, exerts this selective effect is unclear, although a more rapid response of memory T cells and accelerated re-expression of interleukin 2 receptors on cells previously activated in vivo have both been suggested.

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The anti-idiotypic network in T cell vaccination

The derivation of protective T cell clones allowed further investigation of the mechanisms involved in TCV. Since protected rats, but not those immunized with control cell lines, manifested delayed-type hypersensitivity against MTb, and since protection could be transferred from A2b-vaccinated to naive rats by inoculation of activated spleen or thymus cells from vaccinated animals (Lider et al, 1987), it appeared likely that protection was T cell mediated. Immunization against the TCR of the vaccine T cells was presumed to be relevant, but the absolute requirement for activation of the vaccinating cells suggested that other molecules must also be important. Using EAE as a model, Lider et al (1988) attempted to elucidate the pertinent factors. Rats were vaccinated in the hindfoot with the MBP-specific en~:ephalitogenic T cell clone Z l a at a dose which did not cause EAE but could generate protection. Draining LN cells and, a few days later, cells from systemic LNs were shown to be able to transfer protection to naive recipients. LN cells were then cultured with irradiated Z l a cells as stimulators in a limiting dilution assay. The LN cells specifically responsive to Z l a fell into two major categories, CD4+ C D 8 - helper T cells and CDS+ C D 4 - suppressor T cells. The helper cells, like MBP itself, stimulated Zla, whilst the CD8+ cells suppressed the response of Z l a to MBP. These results suggested that TCV mediated its effect by inducing an anti-idiotypic network. It appears most likely that the network is created by the TCR of Z l a since this, the receptor for the MBP antigen, is what differentiates Z l a from other cells to which the network cells do not react. Since TCV responses are elicited with a very low dose of cells, it seems probable that the response is a recall of pre-existing reactivity to anti-MBP T cells rather than a primary response generated de novo. The contribution of the suppressor CD8 cells to the protection afforded by TCV is self-evident since they suppress the encephalitogenic Z l a clone. The role of the CD4 helper cells, which in vitro actually stimulate Zla, is less obvious. However, it may be supposed that, unlike their action in vitro, they may aid in vivo resistance either by stimulating delayed-type hypersensitivity responses or by driving the idiotypic network to increase suppression. The role of CD8+ T cells in suppression of EAE has been confirmed by other workers (Sun et al, 1988) using an encephalitogenic MBP-specific T cell line, S1. These authors isolated T cells from the spleens of rats who had recovered from Sl-induced EAE and cultured them with $1, demonstrating a good proliferative response which was not materially enhanced by the addition of MBP. Lymphoblasts obtained by stimulating the splenic T cells with $1 were then expanded with interleukin 2, leading to the generation of CD8+ oLI3T cells which responded to $1 but not to control cells or MBP. These anti-S1 T cells selectively lyse the $1 cell line in vitro and also neutralize its encephalitogenic activity in vivo. Again this appeared to be an anti-idiotypic (anti-TCR) response. Evidence for an anti-idiotypic network (Cohen, 1991a) has also been obtained in AA by studying a newly developed HSP 65-specific T cell clone, M1, which is also an effective T cell vaccine but recognizes a different

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peptide to A2b. Interestingly it also recognizes serf, but in the form of a mammalian acute phase protein not mammalian HSP. Immunization of rats with MTb induced, within a few days, a strong anti-M1 response, suggesting pre-existing immunity to M1. As would be expected in a preformed network, the appearance of the anti-idiotypic response to M1 preceded the development of the idiotypic T cells responding to HSP 65; that is, the regulatory anti-M1 cells developed before the arthritogenic anti-HSP 65 T cells. TCV with clone M1 amplified this preformed network leading to AA. The degree of anti-M1 response was inversely correlated to the severity of the ensuing arthritis, suggesting that this anti-M1 response could downregulate the immune process leading to arthritis. The anti-M1 response was much lower in rats kept in germ-free conditions, suggesting that the anti-M1 idiotypic network is developed in response to environmental bacteria. How does this network relate to TCV? Clone M1 acts as an effective vaccine against AA since rats immunized with M1 were resistant to the induction of AA, and this was associated with enhanced anti-M1 activity. Surprisingly TCV did not simply suppress the response to HSP 65 but altered its kinetics to cause transient enhancement followed by down-regulation. That is to say, vaccinated animals showed an early response to HSP 65 which was then down-regulated by the time arthritis would normally have developed. How can this be explained? In EAE (Lider et al, 1988), as described above, the anti-idiotypic T cells have been shown to be of two types, CD4+ and CD8+. It is thus possible that the initial enhancement of the anti-idiotypic response is due to the CD4+ cells (which would thus contribute to the immunological dominance of HSP 65), and the later down-regulation would be postulated to be due to the CD8+ cells. The anti-ergotypic response in T cell vaccination Although the data implicating an anti-idiotypic response against the TCR in TCV seem certain, they do not explain the requirement that for successful vaccination the vaccine T cells must be activated. Furthermore, although the protection afforded by TCV is in large measure disease specific, as discussed at length above, a much lower degree of protection can also be engendered by vaccination with syngeneic clones of an irrelevant specificity (Lider et al, 1987). Lohse et al (1989) attempted to address this very question by identifying a group of T cells which can control EAE despite recognizing not the TCR but a surface marker on activated T cells. These T cells, termed anti-ergotypic T cells, were generated by immunizing rats with activated syngeneic T cell clones which were not MBP specific and therefore lacked the relevant TCR. Anti-ergotypic T cells could also be detected in vivo after administration of antigen, suggesting their physiological relevance. These anti-ergotypic T cells were shown to protect against EAE whether the disease was induced by MBP itself or by adoptive transfer of MBP-specific T cells. In terms of mechanism, the anti-ergotypic cells appeared to be both CD4+ and CD8+, and their in vitro response was stimulated by the membrane fraction rather than the supernatant of activated T cells. Furthermore, the anti-ergotypic response is not restricted by MHC class I or class II

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antigens in vivo or in vitro. Taken together, the data support the concept that, whilst the primary protective component of TCV is an anti-idiotypic response directed against the TCR, there is also a secondary element involving the recognition of as yet undefined structures on activated T cells, termed the anti-ergotypic response. T cell vaccination in spontaneous animal autoimmune disease

All the animal diseases so far considered are artificial models elicited by the immunization of animals with bacterial or tissue antigens. It was conceivable that T cell vaccination is only effective in this artificial situation. Thus, it is particularly important that TCV has also been shown to be beneficial in a spontaneous animal autoimmune disease, insulin-dependent diabetes mellitus (IDDM) in the NOD mouse (Cohen, 1991a). The disease begins at 4--6 weeks of age as a mononuclear cell infiltration of the pancreatic islet cells which gradually destroys the insulin-producing pancreatic beta cells. At this stage the insulitis is subclinical; overt hyperglycaemia develops only at about 4-6 months of age, when the remaining beta cells produce insufficient insulin to maintain glucose homeostasis. Like other animal autoimmune diseases, the beta cell destruction appears to be mediated by autoimmune T cells (Bendelac et al, 1987). Recently, the causative antigen has been identified as the mammalian 65kDa (Cohen, 1991a; Elias et al, 1991). As IDDM develops, an HSP 65 cross-reactive antigen appears in the blood, followed by mammalian HSP 65-reactive T cells and then antibodies. These T cells are not simply a consequence of the destructive process, since C9, a 65 kDareactive clone derived from them, is able to mediate severe disease within 1 week of transfer into prediabetic NOD mice or the histocompatible NON.H-2 N~ strain which does not spontaneously develop diabetes. Immunization with HSP 65 induced the disease in NOD and NON.H-2 N~ mice. NOD mice, vaccinated with irradiated T cells from the C9 clone or with anti-HSP 65 T cell blasts, were shown to be protected against IDDM (Cohen, 1991a). Interestingly, in terms of the mechanism of T cell vaccination, the inhibition of the diabetes was preceded by a down-regulation of anti-HSP 65 immunity in the mice. Neither protection from the immunological disease nor an alteration in anti-HSP 65 immunity was seen in mice immunized with control clones. There is also evidence of a pre-existing anti-idiotypic network in diabetes in NOD mice (Cohen, 1991a). A T cell response to the HSP 65 kDa diseaseinducing C9 clone can be demonstrated prior to the development of diabetes. Since diabetes in NOD mice arises spontaneously, unlike AA, this anti-C9 response develops without HSP 65 immunization. Similar to AA, the strength of this anti-idiotypic network, as estimated by the level of anti-C9 response, influences the clinical expression of the disease. Indeed, in male mice, who do not develop diabetes, the anti-C9 T cell response remains persistently high, whilst in female mice it falls just prior to the development of the disease. TCV with anti-HSP 65 cells enhances this natural antiidiotypic network and aborts the development of diabetes in susceptible animals.

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Summary TCV was initially developed in artificial animal models of autoimmune disease such as AA and EAE. It was subsequently also shown to be effective in the spontaneous autoimmune animal disease, diabetes in the NOD mouse. Initial work demonstrated that, like other forms of vaccination, TCV could protect against disease, but further study showed that TCV could also down-regulate an ongoing T cell-mediated immune process, thus being effective in the treatment of established disease. TCV is effective in autoimmune diseases mediated by T cells. In TCV, a T cell line or clone known to induce the disease is attenuated (by a variety of physical or chemical means) and injected into the animal either prior to disease induction (for protection) or after disease onset (for treatment). An immune response is induced against the vaccinating T cell, and hence against the disease-inducing autoimmune T cells from which the vaccine was made. This response is mainly anti-idiotypic (anti-TCR), but also involves other elements, for example an anti-ergotypic response. The immune response comprises both CD4+ cells, which may be responsible for a transient initial enhancement in the disease-inducing T cell response, and CD8+ T cells, which are probably the main element leading to suppression and cytotoxicity against the autoimmune disease-inducing T cells. Of great interest, it is now becoming clear that TCV does not induce de n o v o this anti-idiotypic immune response but, rather, amplifies a pre-existing natural immunoregulatory network. Finally, it has been shown that TCV is also effective when, instead of pathogenic lines and clones, attenuated unseparated LN cells from primed animals are used to make the vaccine, providing they are activated. This is of great relevance in the potential application of TCV to human autoimmune disease since in humans the pathogenic T cells cannot be precisely identified.

TCR PEPTIDE VACCINATION IN ANIMAL MODELS Once it was clearly established that the major T cell molecule determining protection in TCV was the TCR, various investigators set out to determine whether proteins from this receptor could be used as vaccines. As well as being of great theoretical interest, since it would confirm the dominant role for the TCR in TCV, the use of TCR peptides as vaccines would have a number of advantages, both theoretical and practical, over TCV and over therapy with anti-TCR monoclonal antibodies.

Effectiveness of TCR peptide vaccination The initial studies were performed in EAE induced in Lewis rats by guinea pig MBP (Vandenbark et al, 1989). In this model, the T cells responding to the major encephalitogenic epitope of MBP (residues 72-89) use the TCR

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genes Vot2 and V[38. A 21 amino acid peptide, predicted to be immunogenic for T cells, was identified within the V138 complementarity determining region 2 (CDR2), which is the hypervariable TCR region involved in binding to the peptide/MHC complex. This peptide, TCR-V[38-39-59, was synthesized together with a homologous sequence from an irrelevant TCR, V1314. Animals vaccinated with the V[38 peptide, but not with the VI314 peptide or saline, were completely protected from subsequent induction of EAE. The absence of protection after V[314 immunization was not due to failure to stimulate a specific immune response, as there was an equivalent T cell and antibody response to both peptides. Interestingly, the V[38-specific T cells expressed CD4 strongly but CD8 only weakly, yet their response to MBP was restricted by MHC class I. In an extension of this work, TCR-V[38-39-59 was demonstrated to protect against EAE, not only if it was given before the induction of E A E as described above, but also if it was given during the induction period prior to the onset of clinical disease. In a similar study, Howell et al (1989) also identified TCR pePtides which could act as vaccines. The restricted TCR repertoire of encephalitogenic T cells is not confined to the Lewis rat. Indeed, despite variation in the MBP peptide recognized and in the MHC restriction, there is preferential usage of VoL8, Va2 and/or Vow4 by such T cells in most rodent species. To select suitable peptides for vaccines, these workers analysed the sequences of TCR genes from encephalitogenic T cells in a variety of EAE models and identified conserved amino acid sequences in the TCR[3 chain VDJ (variable-diversity-junctional) region and in the Ja elements. On the assumption that such conserved idi0topes could be implicated in E A E pathogenesis, they vaccinated animals with synthetic peptides corresponding to these conserved sequences and were able to demonstrate protection against E A E although different peptides possessed different protective capacities. Mechanism of TCR peptide vaccination

The mechanism by which TCR peptide vaccination provided protection was extensively investigated in the study by Vandenbark et al (1989). Protection was T cell-mediated since V[38-specific T cells, isolated from the LNs of protected rats, conferred passive protection against EAE in naive rats. It was EAE specific since recognition of other antigens was unimpaired. It was also not due to deletion of encephalitogenic T cell precursors since MBPspecific T cell lines could be derived from protected animals and these were encephalitogenic in naive animals. Furthermore, although the V[38-specific T cell line proliferated to V[38+ T cells in the absence of accessory cells, indicating direct recognition of the TCR peptide on the cell surface, the line was not cytotoxic for MBP-reactive T cells. The major mechanism of protection by MBP-specific T cells appeared to be deviation of the MBPspecific T cell response away from the encephalitogenic 72-89 sequence towards other epitopes, for example the normally poorly recognized 87-99 peptide. Antibodies specific for TCR-V[38-39-59 were also shown to play a part in the protective role of the peptide. It remains unclear whether the V[38

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specific immune response is directed against the natural TCR or, perhaps more likely, an MHC-associated processed fragment of the V[38 chain. In the study by Howell et al (1989), the protective mechanism was not analysed in detail. However, the peptide which was found to be most protective was shown to stimulate anti-S1, a cytotoxic T cell line shown previously (Sun et al, 1988) to recognize anti-idiotypic determinants on the $1 encephalitogenic T cell line, as outlined above. TCR peptide vaccination in established disease The demonstration that short TCR peptides can protect against the induction of autoimmune disease not only supports the concept that T cell recognition of the TCR is involved in the protective effect of TCV, but also provides an alternative way of inducing such anti-idiotypic immunity, provided always that the pathogenic T cells express a limited TCR repertoire. However, protection is of little relevance to human autoimmunity, where the most important issue is the therapy of established disease. Can TCR peptides, like TCV, be used in this way? Offner et al (1991) compared treatment with the TCR-V[38-39-59 peptide and the irrelevant V[314 homologue in rats with clinical EAE. Whether administered intradermally, subcutaneously or with CFA, TCR-V[38-39-59 was able to arrest disease progression within 24 h and, over the next 48 h, to effect a virtually complete recovery. The average recovery time in control rats was 6.5 days, whereas in the treated group it was halved to 3.5 days. Pre-existing immunity to TCR peptides The rapid response to TCR-V[38-39-59 suggested a recall response, implying that rats with EAE have immunity to the peptide prior to vaccination. In support of this idea, rats with EAE, but not naive or CFA-injected rats, were shown to develop significant delayed-type hypersensitivity responses after intradermal injection with the TCR-V[38 but not the V[314 peptide (Offner et al, 1991). Further analysis of the immune response to TCRV[38-39-59 was undertaken in MBP-immunized but non-TCR vaccinated animals just before they developed clinical EAE (Offner et al, 1991). These animals, unlike naive and CFA-injected rats, had vigorous T cell and antibody responses against TCR-V~8-39-59. However, despite this, the rats still progressed to clinical EAE, indicating that the degree of immunity was insufficient to prevent the disease. After undergoing a natural recovery from EAE, these rats continued to display both cellular and humoral immune responses to the TCR-Vt38-39-59 peptide, but the response was only a fraction of that of animals in whom MBP-induced EAE had been treated with TCR peptide vaccination. Consistent with previous experiments (Vandenbark et al, 1989), both naturally recovered and TCR-V[38 peptide treated animals displayed immunity to the encephalitogenic MBP peptide, indicating that treatment led to regulation and not deletion of the encephalitogenic T cell specificities.

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Summary Taken together, these results support the concept that expansion of the encephalitogenic (V[~8+) T cells during EAE leads to the development of a regulatory T cell and antibody response directed, at least partially, against the TCR-V[38-39-59 peptide. Immunization with the TCR-V68 peptide, by triggering this pre-existing anti-idiotypic network, amplifies the regulatory response and produces clinical benefit both in terms of protecting against E A E and treating established disease. As discussed above, other TCRderived peptides can also induce protective immune responses. The relative importance of immunity to the various TCR peptides, and also the importance of anti-idiotypic (anti-TCR) immune responses, compared with other regulatory mechanisms in the regulation of EAE remains unclear. Nonetheless, vaccination with TCR peptides manifestly provides an effective means of protection and therapy in this disease. Confirmatory studies in other animal models are awaited.

COMPARISON OF WHOLE T CELL AND TCR PEPTIDE VACCINATION What are the maj or advantages of TCR peptide therapy, compared with other treatment such as TCV or TCR-specific monoclonal antibodies, as a remedy for autoimmune disease? First, the efficiency of whole TCV is variable, perhaps relating to the difficulty of ensuring a standard preparation. Second, whole TCV induces reactivity against determinants other than the TCR. Although some of these, for example the anti-ergotypic response (Lohse et al, 1989), may be relevant to protection, others may be irrelevant or even harmful. Third, in an outbred population like humans, T cells used as vaccines must be autologous to avoid an alloreactive response against the vaccine. This means that an individual preparation must be made for each patient, a highly labour-intensive procedure which is unlikely to be practical on a large scale. In contrast, if TCR peptides were effective as vaccines, they could be manufactured as standard preparations. The evidence from Vandenbark and his colleagues (Vandenbark et al, 1989; Offner et al, 1991) that TCR peptide therapy is effective even when vaccination is performed in the absence of adjuvant is also important in considering the treatment for human disease. Nevertheless, there is one essential requirement which must be fulfilled if peptides from the TCR are to be used as vaccines: the disease-inducing T cells must use a limited repertoire of TCR V region genes. The evidence for and against this notion in human disease will be further discussed below. If there were such a restricted V gene usage, TCR peptide vaccination would have some clear advantages over the alternative technique of deletion of the offending T cell clone with anti-TCR monoclonal antibodies. First, unlike antibody, synthetic TCR peptides do not contain foreign material and, second, TCR peptide vaccination would induce a persistent active immune response, in contrast to anti-TCR monoclonal antibody deletion, where re-evolution of the pathogenic clone from precursors would be a possibility.

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T CELL VACCINATION IN HUMAN AUTOIMMUNE DISEASE Once the effectiveness of TCV had been demonstrated in a variety of animal models of autoimmune disease, investigators were keen to examine its potential in treating the vast number of human autoimmune diseases such as RA and MS in which neither the causative antigen nor the pathogenic T cells are known. As discussed earlier, TCV is of particular interest in this situation since it may offer a therapy which is potentially specific for the immunological process involved in the disease, unlike for example cyct0sporin, anti-CD4 antibodies or inhibition of cytokine pathways. Furthermore, it may be possible to use it in the absence of identification of either the disease-inducing T cells, unlike anti-TCR monoclonal antibody therapy, or the causative antigen, unlike antigen-specific tolerance.

Problems with human T cell vaccination

In attempting to transfer the animal experience to humans we face several seemingly intractable problems (Cohen, 1991b). First, from what cells should the vaccine be derived? In diseases such as RA and MS, because the causative antigen has not been identified and transfer experiments are impossible in humans, no specific T cell lines and clones mediating disease are available to use as vaccines. Furthermore, although LN cells from primed animals have been used as vaccines in animal models, these too are not readily available in patients. Most human studies have been done using T cells from the site of pathology (cerebrospinal fluid in MS and synovial fluid in RA) as a source on the basis that the pathogenic T cells will be at the highest frequency there. However, using limiting dilution analysis in forms of arthritis such as reactive arthritis where the inciting antigen is known (Panayi et al, 1992), most synovial T cells have been shown to be unresponsive to the causative agent and therefore irrelevant to the induction of the disease. Even more importantly, within the lesion there may be regulatory T cells trying to control the immune process, whose down-regulation would be a positive disadvantage. Second, how should the potential vaccine T cells be selected from these various sources and then expanded in the absence of the causative antigen? Using TCR V gene analysis, T cells could be selected on the basis that they came from predominant clones in the synovium. At present there is conflicting evidence (Paliard et al, 1991; Uematsu et al, 1991; Bowness and Bell, 1992) as to whether such a predominance occurs; even if restricted TCR usage were identified, it need not represent expansion of the antigen-specific T cells which mediate the disease. Predominant 'clones' could also represent regulatory antigen-specific T cells or cells which were recruited into the lesion as a result of other irrelevant processes (Panayi et al, 1992). An alternative method of selection is that used for the LN-derived T cell vaccines in animals, where it was shown (Mor et al, 1990) that expansion with concanavalin A, but not interleukin 2 alone, could enrich the frequency of the relevant antigen-specific T cells, presumably because these cells have

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an advantage in culture. Such a technique could be used in humans, although it would be impossible to confirm that concanavalin A had this effect in the human situation, since the disease-related autoantigens are unknown. Another possibility which has been used is to expand the cells with candidate antigens such as MTb 65 kDa HSP in human RA. It now seems unlikely that this is a true causative antigen in RA (Life et al, 1991), and no other potential antigen has yet emerged. Third, how should the T cell vaccine be prepared? Preparation of the vaccine requires two main processes, activation and then attenuation. Activation in animal TCV has been done mainly with the specific antigen, which is not available in humans. Thus activation has been done with mitogens such as concanavalin A, phytohaemagglutinin (PHA) or anti-CD3 antibodies, though caution needs to be exercised to avoitt carry-over of these toxic substances into the preparation to be given to the patient. Clearly there is also no specificity in such a method of activation. Attenuation has been done with a variety of chemical and physical processes in the animal models and in some cases certain techniques are more effective than others; their effect on human T cell vaccines is unknown. Fourth, there has been no toxicity associated with the use of animal TCV but, of course, these have been vaccines of known specificity. Unexpected results could arise in humans, including the unintentional inclusion within the vaccine of the very regulatory T cells which should be enhanced, thus stimulating a suppressive immune response against them. Finally, there is the question of how to assess the effectiveness of the vaccine. Assessment of clinical efficacy presents many difficulties. As with all new therapies, it must be tried initially in those patients for whom conventional treatment has failed. Unfortunately, many of these patients have secondary inflammatory and degenerative processes which are in part responsible for their symptoms; the situation is further complicated by the existence in many human autoimmune diseases of spontaneous relapses and remissions. Immunological assessment is also complex. Since the causative antigen and disease-mediating T cells are unknown, detailed analysis of the anti-idiotypic network and the effect of TCV on it, such as could be carried out in the animal diseases, is impossible. Nevertheless, relevant analyses can be performed, including T cell responses pre- and post-vaccination, particularly using the vaccine as a stimulus but also more generally to mitogen and recall antigen. Despite these multiple problems, the prospect of a disease-specific therapy in currently untreatable chronic progressive conditions led inexorably towards a trial of TCV in human diseases such as RA and MS. T cell vaccination studies in human disease

The earliest human studies of TCV were performed by Hailer and Weiner in Boston in patients with chronic progressive MS using as vaccines autologous attenuated cerebrospinal fluid T cell clones raised non-specifically (Anonymous, 1991). Whilst clear-cut clinical results could not be expected in a pilot study of such a variable condition, there was some evidence of immunological

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suppression, notably in decreasing the response to PHA. However the expected T cell response against the vaccinating cells proved difficult to demonstrate. Most importantly, the procedure proved entirely without side-effects. In RA, TCV studies have been carried out by three European groups in Mainz, Germany (A. Lohse), in Leiden, Netherlands (F. C. Breedveld and R. R. P. DeVries) and at Guy's Hospital in London (G. H. Kingsley and G. S. Panayi). Again these have been small-scale studies, uncontrolled except for the London study, but some useful information has emerged and the procedure has proved safe in all patients (van Laar et al, 1991). The protocol for the studies was similar in each centre. The vaccine was made, in all but one case where blood lymphocytes were used, from synovial fluid mononuclear cells which were expanded with a mitogen (anti-CD3 antibody or PHA) and interleukin 2 until sufficient cells were obtained. The cells were then activated, attenuated with paraformaldehyde or glutaraldehyde and frozen in aliquots of 50 x 106 for subsequent use. In some patients treated in Leiden, vaccination was performed with synovial T cell clones specific for the 65 kDa HSP because of the aetiological role proposed at the time for that protein in RA. The results of these patients did not differ from the others. The patients were given one, two or three vaccinations of 50 x 106 T cells, either intradermally or subcutaneously, spaced approximately 1 month apart. They were followed both clinically, using standard clinical and laboratory measures of disease activity, and immunologically, analysing T cell subsets, rheumatoid factor, T cell proliferative responses to mitogens and antigens and, most interestingly, the T cell response to the vaccine cells. To date no objective evidence of clinical efficacy has been obtained, although a few patients have reported subjective improvement. No major changes in T cell subsets have been documented. In patients who were positive for immunoglobulin G rheumatoid factor the titre fell after vaccination, but the effect on T cell proliferation to mitogens and antigens was variable. T cell proliferation to the vaccine preparation, the 'antivaccine response', was demonstrated in some patients but not others. There are many reasons for these, at first sight disheartening, results. As discussed in the previous section, TCV is a complex process and it has not been possible exactly to reproduce in humans the technique used successfully in animals. Major differences include the source of the T cells, which in humans has been from the site of inflammation rather than from the blood or LN as in the animal studies. The preparation Of the vaccine, in terms of expansion, activation and attenuation, also differs from the rodent studies; in any case, it is unclear how parallel animal and human responses to these processes are. A second major problematic area is the difficulty of assessment of improvement in diseases such as MS and RA. In these conditions spontaneous relapses and remissions occur; furthermore, many of the patients treated with biological agents such as TCV have long-standing and resistant disease, often with irreversible end-organ damage, unlikely to respond to such therapy. Most problematical of all, since RA and MS are diseases caused by currently unidentified antigens, it is also not possible to measure down-regulation of the pathogenic process by TCV.

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Future directions

As a result of these studies, two new areas of investigation are underway. First, work has begun on TCV in primates, which may enable some of the remaining questions to be answered in a species more similar to man. Second, studies in TCV in the immediate future are likely to involve diseases where the antigen is known or where there are strong candidate antigens. For example, further work in MS is likely to utilize vaccines made of MBP-specific T cell lines or clones since there is now evidence to support an aetiological role for MBP in MS (Ota et al, 1990). Information from such work can then be applied to diseases such as RA.

TCR PEPTIDE VACCINATION IN HUMAN DISE~.SE

The application of TCR peptide vaccination to human disease is at an even earlier stage than human TCV. This is primarily because this technique cannot be used unless preferential usage of a particular TCR V region gene by the disease-causing cells can be demonstrated. To date, despite a considerable amount of work, there is no consensus on whether a limited TCR repertoire exists either in R A (Paliard et al, 1991; Uematsu et al, 1991; Bowness and Bell, 1992) or in MS (Steinman et al, 1992). It is even less clear if any restriction in TCR usage is patient or disease specific or which TCR V regions should be used as vaccines. Nevertheless, despite these formidable problems, A. A. Vandenbark and colleagues have begun to use TCR peptides in therapy on an individual patient basis, selecting the TCR Vf3 chain from which to derive the vaccine peptide by analysing TCR usage in cerebrospinal fluid cells from each patient. No results are yet available.

SUMMARY

Current theories of the aetiology of R A point to a central role for the trimolecular complex comprising the M H C class II molecule on the surface of the APC, the antigenic peptide and the TCR on the disease-inducing T cell. Thus the arthritogenic T cell is an important target for new therapy. However, it cannot be directly identified because the causative antigen is unknown, so indirect techniques such as TCV and TCR peptide vaccination are required. In TCV, T cells thought to mediate the disease, in an activated and attenuated form, are injected into the patient, who then develops a specific immune response against these pathogenic T cells. TCV has been shown to be effective in protecting against and treating a variety of animal models of autoimmune disease, including A A , E A E and I D D M in NOD mice. The vaccines initially comprised clones and lines of T Cells shown to be capable of transferring the disease, but later unseparated LN cells were also shown to be effective, paralleling more closely the human situation. Interestingly, it has become clear that TCV does not create its own regulatory network but

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amplifies a n a t u r a l i m m u n o r e g u l a t o r y n e t w o r k which f o r m s as t h e d i s e a s e d e v e l o p s . T h e m a j o r s t i m u l a t i n g m o i e t y o n t h e v a c c i n a t i n g T cell is its r e c e p t o r ( a n t i - i d i o t y p i c r e s p o n s e ) , a l t h o u g h t h e r e is also an a n t i - e r g o t y p i c ( a n t i - a c t i v a t e d T cell) r e s p o n s e . F o r this r e a s o n t h e t e c h n i q u e of T C R p e p t i d e v a c c i n a t i o n was d e v e l o p e d , w h i c h utilizes o n l y a s h o r t p e p t i d e f r o m t h e T C R o f t h e d i s e a s e - c a u s i n g cells to s t i m u l a t e an i m m u n e r e s p o n s e a g a i n s t t h e m . This is effective in t h e p r e v e n t i o n a n d t r e a t m e n t o f E A E , w h e r e t h e r e is a p r e f e r e n t i a l u s a g e o f TCR-V[38 b y e n c e p h a l i t o g e n i c T cells. T h e a p p l i c a t i o n o f b o t h t h e s e t e c h n i q u e s to h u m a n a u t o i m m u n e d i s e a s e is in its infancy. S t u d i e s of T C V in M S a n d R A h a v e n o t s h o w n c l e a r - c u t clinical b e n e f i t , a l t h o u g h i m m u n o l o g i c a l c h a n g e s h a v e b e e n o b s e r v e d ; c o m p a r i s o n of m e t h o d o l o g y with t h e a n i m a l w o r k a n d a s s e s s m e n t of results a r e c o m p l e x a n d f u r t h e r studies a r e in p r o g r e s s . S t u d i e s of T C R p e p t i d e v a c c i n a t i o n in M S a n d R A a r e h a n d i c a p p e d b y t h e l a c k o f a c o n s e n s u s o n T C R u s a g e in t h e s e c o n d i t i o n s , b u t a l i m i t e d s t u d y is u n d e r w a y in MS.

Acknowledgements The authors would like to acknowledge the Arthritis and Rheumatism Council for financial support.

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