Springer Semin Immunopathol (1991) 13:99-113

SpringerSeminars in Immunopathology 9 Springer-Verlag 1991

The 65-kDa heat-shock protein in the pathogenesis, prevention and therapy of autoimmune arthritis and diabetes mellitus in rats and mice Ulrich Feige 1 and lrun R. Cohen 2 Department of Inflammation, Pharmaceuticals Research Division, Ciba-Geigy Ltd., R-1056.125, CH-4002 Basle, Switzerland 2 Department of Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel

Introduction

The 65-kDa heat-shock protein (hsp65) is one of the most highly conserved molecules in biological evolution: the hsp65 molecules of prokaryotes and humans are about 50% identical in their amino acid sequence [33, 37]. This remarkable degree of conservation indicates that hsp65 is likely to be of universal importance in cellular life. The function of hsp65 appears to be that of a molecular chaperone involved in the folding and assembly of proteins [23]. This function is acutely important in stress [42] and thus hsp65 has been called a chaperonin as well as a stress protein [23, 33, 42]. The subject of this volume is not the physiological function of hsp molecules but rather the way the immune system relates to hsp molecules. In other words, we look here at hsp molecules as targets of immunity and not as independent agents. The immunological interest in hsp65 molecules stems from three recently discovered facts: (1) hsp molecules of microbes are dominant antigens in the immune response to infections agents; (2) hsp molecules are targets of immune reactions in autoimmune diseases; and (3) hsp molecules are recognized by the immune systems of healthy individuals who are apparently free of active infection or of autoimmune disease. These observations are paradoxical: if an antigen is half-self, why and how should it attract a dominant immune response in infection? If an antigen is associated with the etiology of autoimmune disease, why and how can healthy individuals respond to it without suffering harm? What distinguishes hsp immunity in the states of health, microbial infection and autoimmune disease? T h e s e questions have motivated much of the current research in hsp65 immunology and many of the chapters in this volume review and discuss the findings. The aim of this chapter is to review information about hsp65 immunity in the autoimmune diseases arthritis and insulin-dependent diabetes mellitus (IDDM) in rats and mice. In particular we shall describe the use of hsp65 and Offprint requests to: U. Feige

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its peptide epitopes in novel immunological therapies. Table 1 is a compilation of the published experiments and serves as a guide for the following paragraphs.

Adjuvant arthritis Adjuvant arthritis (AA) can be induced in susceptible strains of rats, e.g., Lewis rats, by heat-killed mycobacteria in oil injected i.d. at the base of the tail or into the foot pad i (for review see [63]). Because of its reproducibility, AA is widely used as an animal model of rheumatoid arthritis (RA), although there are differences between the two diseases [34, 63]. Characteristically paw swelling is found starting 10 to 12 days after injection of mycobacteria. Weight loss is observed in rats with AA and the underlying changes in bone (metaphyseal osteomyelitis, ossifying periostitis, etc.) are evident histologically and radiologically. With progression of disease the edematous paw swelling subsides, but joint destruction and new bone formation along the tarsal and metatarsal bones continues [63]. Clinically important drugs in RA such as non-steroidal anti-inflammatory drugs, steroids, methotrexate, and cyclosporin A inhibit this experimental autoimmune arthritis [3, 63].

Induction and prevention of AA with T cell lines and clones As early as the 1960's it was shown that AA can be transferred to recipients with T cells obtained from Mycobacterium tuberculosis (Mt)-immunized donors at defined times [24, 25, 51, 62]. Later it was found that AA could be transferred more easily if the T cells were restimulated (activated) in vitro with concanavalin A (Con A) before injection into recipients [49]. Cohen and his associates [2, 28] showed that autoimmune diseases could be induced in naive recipients with T cell lines and clones which had been maintained in culture in vitro by biweekly cycles of restimulation with antigen and expansion in interleukin-2 (IL-2). The T cell line A2 was found to induce arthritis in irradiated Lewis rats [28]. Surprisingly, subclones of line A2 were found to either induce arthritis (clone A2b) or to prevent or therapeutically reduce AA (clone A2c or attenuated clone A2b) [19, 29, 39]. The latter has been termed Tcell vaccination [2, 11, 13, 17, 28]. T cell vaccination experiments have indicated that prevention and therapy of autoimmune diseases in experimental models can be achieved by induction of an anti-T cell immune response (see below). T cell clones which induce disease and/or prove useful for T cell vaccination are valuable tools for the identification of the target antigen(s) and epitope(s) in autoimmune diseases [22, 55, 58, 65, 70].

hsp65 and peptide 180-188 in AA With the development of T cell clones A2b and A2c (see above) it became possible, using them in proliferation assays in vitro, to search for the mycobacterial antigen and epitope responsible for AA [58]. In a series of studies Van Eden and colleagues 1The day of induction of arthritis (e.g., of AA with mycobacteria)is always designatedDay 0

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[55, 58] showed that the driving antigen for T cell clones A2b and A2c was the hsp65 molecule ofMycobacterium bovis. T cell clones A2b and A2c also responded to cartilage proteoglycan in vitro [60], pointing to molecular mimicry as one possible explanation for AA [9, 57, 59, 60]. Surprisingly for both A2b and A2c, the epitope on the mycobacterial hsp65 was found to be identical and could be defined as the nonapeptide sequence 180-188 [58], and refined somewhat later as the heptapeptide sequence 180-186 of mycobacterial hsp65 [55]. With the new information that both the a and /3 chains of the T cell receptors (TcR) of A2b and A2c are identical, it is now no longer surprising that these clones see an identical epitope [56]. Before considering vaccination or therapy based on the use of an antigen or epitope, the question whether the antigen or epitope used has the potential of inducing or exacerbating disease must be studied thoroughly. In mouse and rat models of encephalomyelitis it has been shown that experimental allergic encephalomyelitis (EAE) can be induced with encephalitogenic peptides [70]. Therefore, as a basis for vaccination and treatment experiments with the hsp65 and peptide 180-188 it was mandatory to test whether these antigens were arthritogenic. Experiments in which peptide 180-188 was injected s.p. (subplantar) or i.d. in oil, according to the regimen by which AA is induced with Mt, never showed any indication of arthritogenicity [66]. Interestingly even the whole hsp65 has been found to be not arthritogenic [4, 58, 60]. There are apparently two prerequisites for arthritogenicity (discussed in [65]):(i) antigenicity, which is fulfilled, and (ii) adjuvanticity, which is not fulfilled, by hsp65 and peptide 180-188. Adjuvanticity in the case of induction of AA would be delivered by the whole mycobacteria. However, it is important to note that the conclusion that hsp65 and peptide 180-188 are not arthritogenic is based on experiments with negative results (no appearance of arthritis) and will hold only as long as nobody is able to show arthritogenicity experimentally. The difference between AA and EAE in terms of induction of disease is that EAE is induced with the encephalitogenic peptide in complete Freund's adjuvant (CFA; providing adjuvanticity) as an emulsion, whereas AA is induced with mycobacteria in oil. To test the arthritogenicity of hsp65 and peptide 180-188 might be difficult, because the adjuvant mixture, Mt in oil, used for the induction of AA is itself arthritogenic, and therefore cannot be used to answer the question. Furthermore, administration of Mt in oil as an emulsion is much less arthritogenic than Mt in oil without emulsification (X.-D. Yang and U. Feige, submitted).

Modulation of AA with mycobacteria, hsp65, and peptide 180-188 Identification of the causative antigen of an autoimmune disease makes it possible to attempt immunotherapy using the antigen. Despite the fact that whole mycobacteria are the active ingredient of CFA, experiments were performed to study the potential of mycobacteria as vaccines in AA [24, 25, 51, 71]. With the knowledge that the hsp65 molecule of mycobacteria and possibly a nonapeptide epitope could be causative for AA [58], studies were performed to make use of this knowledge for prevention and treatment of AA [66, 67].

hsp65 (day - 5)

hsp65 (week - 4 to - 3 )

Lewis rat

Lewis rat

Induced by cell walls of streptococci

Induced by CP-20961 - optimal dose - suboptimal dose

CP-20961 arthritis

Peptide 180-188 (days - 3 5 , - 2 0 , - 5 ) (day - 2 0 ) (days - 2 , - 1, - 0 ) (days 7, 8, 9, 10)

hsp65 (week - 4 to - 3 )

Mycobacteria (twice weekly from week - 3 ) (week - 4 ) (day - 3 5 ) (days - 7 , - 5, - 2 )

SCW arthritis

Lewis rat

Induced by mycobacteria

Treatment with

AA

Species

Spontaneous or induced

Autoimmune disease

IFA/i. d.

IFA/i. p.

Oil/i.p. Oil/i. p. Oil/i. p. Oil/i. p.

Oil/i. p. IFA/i. d. Saline/i. p.

Saline/i. p. Oil/i. d. Oil/i. p. Orally

Adjuvant/ route

Slight suppression Prevention

[53, 54]

Prevention

Table 1. continued next page

[4]

[67] [66] [66] [66]

[58] [4] [4]

[71]

b

[25] [24, 51]

Reference

Prevention/suppression Slight suppression Slight suppression Slight suppression

Prevention/suppression Prevention No effect

Prevention/suppression Prevention/suppression Prevention No effect

Effect on autoimmune diseasea

Table. 1. Involvement of the 65-kDa heat-shock protein (hsp65) in several autoimmune diseases in animals

O

O

to

Induced by type II collagen

"

Induced by pristane

Spontaneous

CIA

CIA

Pristane arthritis

IDDM

NOD mouse

CBA/Igb mouse

B10.RII1 mouse

Lewis rat

Species

p277 (at 4 to 5 weeks of age)

hsp65 (at 5 weeks of age)

Mycobacteria (at 5 weeks of age)

hsp65 (day - 10)

Induction of transient diabetes, thereafter protection from spontaneous diabetes Protection from spontaneous diabetes Protection from spontaneous diabetes, and from hsp65-induced diabetes

PBS/i. p.

IFA/i. p.

Protection from spontaneous diabetes

Prevention

Slight suppression

No effect

Slight suppression

Effect on autoimmune disease a

IFA/i. p.

CFA/i. d.

IFA/i. p.

IFA/I. p.

Oil/i. p.

Peptide 180-188 (days - 3 5 , - 2 0 , - 5 ) hsp65 (week - 1)

IFA/i. d.

Adjuvant/ route

hsp65 (week - 4 to - 3 )

Treatment with

[22]

[21]

[21]

[47]

[50]

[32]

[4]

Reference

a Effects of treatment are given as 'prevention'/'protection' if there is not disease at all, or as '(slight) suppression' if there is (slightly) reduced disease b X.-D. Yang and U. Feige, submitted AA: Adjuvant arthritis; SCW: streptococcal cell wall; CP-20961: synthetic adjuvant [N, N-dioctadecyl-N', N'-bis (2-hydroxyethyl) propanediamine]; CIA: type II collagen-induced arthritis; pristane: 2, 6, 10, 14 - tetramethylpentadecane; IDDM: insulin-dependent diabetes mellitus; IFA: incomplete Freund's adjuvant; CFA: complete Freund's adjuvant; NOD: non-obese diabetic

Spontaneous or induced

Autoillunune disease

3.

,..r

d,

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Vaccination against AA with Mycobacteria. Soon after the first description of AA in rats attempts were made to prevent or modulate disease in an antigen-specific way. At that time low- and high-zone tolerance was extensively discussed [30]. Rats were pretreated with mycobacteria before the arthritogenic challenge with mycobacteria (Table 1). The very first reports showed that it takes about 4 weeks to establish resistance to a later arthritogenic challenge with mycobacteria [24, 25, 51]. In an elegant study using cell-transfer experiments it was shown that the resistance to AA induced by i.p. pretreatments twice weekly for 3 weeks with mycobacteria in saline can be transferred with lymph node cells to recipients [25], and further that this resistance to AA is due to the inhibition of the induction but not the effector phase of AA; inhibition of active AA but not passively induced AA was seen [25]. Interestingly, it was found that pretreated donors, as well as recipients of their cells, developed tuberculin reactions comparable to those of AA controls [25]. These findings suggested that induction of tolerance was not the underlying mechanism of resistance to AA. Moreover, it was recently reported that oral induction of tolerance to Mt was without effect on incidence, day of onset, and severity of subsequent AA [71]. Recently it was investigated in more detail whether the resistance to AA in rats pretreated with Mt, was due to a suppression of the cellular immune response to mycobacterial antigens. Thirty-five days after the arthritogenic challenge with mycobacteria enhanced responses to purified protein derivative (PPD) and peptide 180-188 were found in delayed-type hypersensitivity (DTH) reactions and in proliferation assays of splenic T cells in mycobacteria-pretreated, AA-resistant rats (X.-D. Yang and U. Feige, submitted). Thus, resistance to AA following treatment with Mt involves not tolerance to Mt but some modification of the immune response (see below). At this point it should be mentioned that there are reports on AA in Fischer rats. This strain is major histocompatibility complex (MHC) class II identical to Lewis rats [27, 56]. Interestingly, rats kept under conventional housing conditions or colonized with E. coli are resistant to AA, whereas germ-free rats are susceptible to AA [35]. Susceptibility or resistance to AA in Fischer rats is believed to be regulated by a cellular immune response specific for hsp65 [27, 56]. Vaccination and treatment of AA with hsp65 and peptide 180-188. With the availability of recombinant mycobacterial hsp65, the effect of specific vaccinations on subsequent AA could be studied (Table 1). The results confirmed that hsp65 plays a crucial role in AA. Rats pretreated with hsp65 3 to 4 weeks before the arthritogenic challenge were completely protected from AA [4]. To achieve the protective effect, hsp65 had to be used in an immunogenic form in oil [4, 58]. Application of the hsp65 in saline i.p. was without effect on the subsequent AA [4]. Low concentrations of hsp65 administered during arthritis resulted not in exacerbation but in an earlier remission of AA (I.R. Cohen, unpublished). The identification of peptide 180-188 as the epitope for T cell clones A2b and A2c [58] triggered investigations on the vaccinative and therapeutic potential of this epitope in AA. As it had been shown that pretreatment with mycobacteria i.p. is fully effective in mediating unresponsiveness to AA only when the

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mycobacteria are applied 4 to 8 weeks prior to induction of AA [51], the effect of pretreatment with peptide 180-188 at days -35, -20, and -5 on subsequent AA was investigated [66]. Pretreatment with 0.1 mg peptide 180-188 in incomplete Freund's adjuvant (IFA) i.p. resulted in complete prevention of AA in 9 out of 16 rats (Table 1). These three pretreatments with peptide 180-188 are neccessary; skipping any one of the pretreatments dramatically diminished the effect (X.-D. Yang and U. Feige, unpublished). In rats pretreated with peptide 180-188 and resistant to AA a cellular immune response to peptide 180-188 was found [67]. This is similar to what has been observed for whole Mt and for hsp65 (see above), again indicating that resistance to arthritis is not due to tolerance to hsp65 or its peptide 180-188 epitope. The antigen specificity of the pretreatment was tested using peptide 181-188 or N-acetylated peptide 181-188. The lack of threonine at position 180 resulted in the loss of the protective capacity ([66, 67] and X.-D. Yang and U. Feige, submitted). T cell clones A2b and A2c also have an absolute requirement for threonine at postion 180 to respond in vitro [55]. Collagen II-induced arthritis (CIA) in rats differs from AA in the immune response found towards Mt and type II collagen [7]. So it was not surprising that pretreatment with peptide 180-188, which leads to resistance to AA, was without effect on CIA (X.-D. Yang and U. Feige, submitted). This supports the idea that pretreatment with peptide 180-188 is not a general immunomodulating treatment. Other treatment schedules were distinctly less effective than the vaccination with three pretreatments with peptide 180-188 described above (Table 1). Schedules chosen were: pretreatment at day - 2 0 (prophylactically), treatment at days - 2 , - 1, 0 (around induction of AA), and at days 7, 8, 9, 10 (just before clinical onset of AA). All the latter treatment schedules reproducibly led to a suppression of AA in terms of clinical scores (10%-30%) and radiographical scores (20%-50%) [66]. None of the above treatment schedules resulted in full suppression of disease. In addition pretreatment with peptide 180-188 at day - 2 0 was effective only when peptide 180-188 was applied i.p. [66]. Application of peptide 180-188 at day - 2 0 i.d. at the base of the tail, in the footpad or i.v. did not have any effect on subsequent AA [66].

Effects of hsp65 in other animal models of arthritis In addition to AA, other forms of experimental arthritis have been shown to be inhibited by pretreatment with hsp65: streptococcal cell wall (SCW) arthritis in rats [53, 54], pristane (2, 6, 10, 14-tetramethylpentadecane)-induced arthritis in mice [50], and to a lesser extent CP-20961 IN, N-dioctadecyl-N', N'-bis (2-hydroxyethyl)-propanediamine]-induced arthritis in rats [4] and CIA in rats [4] and mice [32]. There would not seem to be any obvious immunological relationship between these other types of arthritis and AA; each type is induced by another, apparently immunologically unique, inducer. Hence, the finding that administration of hsp65 can inhibit each type of arthritis is intriguing. It is conceivable that each of the different types of induction of arthritis involves immunity to hsp65 at some common point in the pathogenesis of arthritis. Various possibilities may

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be considered and comparative studies of the pathophysiology of these types of arthritis will be required to clarify the issue. Are all the types of arthritis controllable by vaccination with the 180-188 peptide or by T cell vaccination with T cells responsive to this peptide or other peptide epitopes of hsp65? Does hsp65 immunity follow treatment with SCW or pristane? If so, to which epitopes? Are similar T cell receptor genes used by the arthritogenic T cells? To illuminate the above questions we might look at some results from different types of arthritis. A very similar disease to AA is induced in Lewis rats by the synthetic adjuvant, CP-20961 [6]. Pretreatment of Lewis rats with hsp65, which induces unresponsiveness to AA, only slightly suppresses CP-20961-incluced arthritis [4], but does prevent CP-20961-induced arthritis if the dose of CP-20961 chosen for the arthritogenic challenge is suboptimal [4]. This illustrates that diseases which have been claimed to be very similar or identical, such as AA and CP-20961-induced arthritis [6], might be distinguished by their differential response to antigen-specific treatment with the same antigen. In pristane arthritis, arthritic mice show a low response of their T cells to hsp65 in vitro [50]. In contrast, pristane-immunized but non-arthritic mice present with a high proliferative T cell response to hsp65 in vitro, as do mice which have been protected from pristane arthritis by hsp65 pretreatment [50]. However, in SCW arthritis in rats the T cell response to hsp65 in vitro is diminished in animals pretreated with hsp65 and resistant to the arthritogenic challenge with SCW [53, 54].

Mechanisms of resistance to arthritis

At this point we have more questions than answers when trying to understand exactly how resistance to arthritis works. It is also fair to say that we know very little about the mechanisms by which arthritis is induced. When we know more clearly how hsp65 immunity is involved in the pathogenesis of arthritis we may have a clearer idea of how treatment with hsp65 or its peptides might actually abort the disease process in AA and in the other types of arthritis. It should be noted that resistance to an arthritogenic challenge following pretreatment with hsp65 might be accompanied by an enhanced (e.g., in pristane arthritis in mice [50]) or a reduced (e.g., in SCW arthritis in rats [53, 54]) T cell response to hsp65 in vitro. Taking stock of the present situation, we can make the following points: 1.An arthritogenic T cell clone, A2b, that sees the peptide 180-188 of mycobacterial hsp65, recognizes a component of the cartilage proteoglycan [57, 59, 60] but does not respond to mammalian hsp65 (N. Karin, D. Markovits and I.R. Cohen, unpublished). There is no homology between the 180-188 sequence of mycobacterial and mammalian hsp65 molecules (see [33, 61]). Therefore, antigenic mimicry between the 180-188 epitope and cartilage are probably involved in AA [57, 60]. Is such mimicry also involved in the other arthritides which respond to pretreatment with hsp65 such as SCW or pristane arthritis? 2. Direct immunization with hsp65 or with its peptide epitopes is not arthritogenic. Therefore, there is no proof that hsp65 immunity is sufficient to cause arthritis.

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3. Nevertheless, a type of hsp65 immunity is apparently required to induce arthritis; disease can be prevented or even treated by hsp65, by the peptide 180-188 or by T cell vaccination with clones that recognize hsp65. 4. Resistance to arthritis is not associated with tolerance or non-responsiveness to hsp65 [65, 66]; rather the kinetics of the response seem to be modified (N. Karin and I.R. Cohen, in preparation). Therefore, resistance to arthritis is not simply due to the lack or depression of hsp65 immunity. 5. T cell vaccination that prevents AA induces augmented anti-idiotypic T cell reactivity to the anti-hsp65 T cells associated with arthritis [40]. Therefore, it is conceivable that arthritis may be regulated by T cell networks 2. Prevention of AA by pretreatment with non-arthritogenic doses of mycobacteria also augments the anti-idiotypic T cells (N. Karin and I.R. Cohen, in preparation). It is not known whether peptide vaccination also activates the anti-idiotypic network. Thus, arthritis and its regulation involve poorly understood antigenic crossreactivities as well as complex cell interactions. Immunity to the hsp65 molecule is somehow a key factor.

hsp65 and diabetes The association of hsp65 immunity with microbial infection and autoimmune arthritis have now been extended to the autoimmune diabetes of the non-obese diabetic (NOD) strain of mice [21, 22]. NOD mice spontaneously develop a form of IDDM very similar to the IDDM that affects humans [20]. The mice develop insulitis at about 1 month of age that progresses to overt hyperglycemia by 4-6 months of age when the remaining/3 cells can no longer supply a sufficient amount of insulin. Elias and her colleagues [21] first reported that the development of insulitis in NOD mice was marked by the spontaneous development of antibodies and T cells reactive to hsp65, and T cell clones reactive to hsp65 transferred diabetes to young pre-diabetic NOD mice. Immunization to hsp65 in adjuvant (oil) induced insulitis and hyperglycemia, and the administration ofhsp65 in a non-immunogenic form (PBS) prevented IDDM [21]. All this was done using mycobacterial hsp65. Subsequent work has shown that the true epitope is in the mammalian hsp65 molecule: reactivity of T cell clones isolated whith mycobacterial hsp65 was found to be fivefold greater to human or mouse hsp65 than it is to mycobacterial hsp65 [22]. The epitope was identified to reside within a 24 amino acid peptide of the human hsp65 sequence (positions 437-460; see [33]), called the p277 epitope [22]. The hsp65 molecule was cloned and sequenced from a mouse/3 cell tumor and the human p277 peptide was found to differ by 1 amino acid from the mouse hsp65 molecule [5]. Moreover, T cells responding to human hsp65 also responded to mouse hsp65. Thus, NOD diabetes is associated with true autoimmunity to mouse hsp65. 2 Additional support for a regulation of AA by T cell networks stems from results with T cell line M1 which recognizes hsp65 but an epitope other than clone A2b. M1 can vaccinate rats against AA as efficiently as clone A2b [11, 14]

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The association between IDDM and hsp65 autoimmunity seems to be critical in the pathogenesis of disease; the reactivity appears spontaneously with the disease process, active immunization to hsp65 causes disease and T cell clones responding to a defined peptide transfer disease. Autoimmunity to hsp65 is not only sufficient to cause IDDM in NOD mice, it is also necessary for the development of spontaneous IDDM. As we shall relate below, reducing or preventing hsp65 autoimmunity aborts the development of IDDM. How hsp65 autoimmune T cells cause diabetes is not entirely clear, but recent evidence shows that the p277 epitope of hsp65 is present in the secretory granules of healthy/3 cells (in preparation). Thus, hsp65 may be involved in the assembly and/or secretion of insulin and this physiological function of hsp65 could specifically mark/3 cells for destruction by anti-hsp65 autoimmunity.

Regulation of hsp65 autoimmunity in IDDM by T cell vaccination As was found in experimentally induced autoimmune disease such as AA, EAE [2, 13, 28] or experimental autoimmune thyroiditis [43], T cell vaccination with T cells reactive to the target antigen could induce resistance to IDDM [22]. In this case the attenuated anti-hsp65 T cells aborted the spontaneous process of hsp65 autoimmunity. Thus, T cell vaccination can be effective in spontaneous autoimmunity as well as in experimentally induced autoimmunity. T cell vaccination also induced resistance to diabetes adoptively transferred by anti-hsp65 T cells or actively induced by immunization with hsp65 in adjuvant [22]. As found in EAE and in AA, T cell vaccination of NOD mice activated T cells responsive to the specific anti-hsp65 T cells causing disease (in preparation). That a T cell clone can vaccinate different individuals against IDDM suggests that the disease is caused by T cells bearing a shared idiotype. Thus, the IDDM of NOD mice may be related to the usage of a restricted set of T cell receptor genes, a finding also seen in EAE [1]. As was observed in Lewis rats [11, 13, 40, 41], NOD mice appear to have a natural network of anti-anti-hsp65 T cells (in preparation). T cell vaccination, thus, augmented the natural regulatory network already present in the animals. The findings of a natural anti-idiotypic network related to hsp65 immunity may be related to the immunological dominance of this antigen [12, 14, 16, 18, 37, 68, 69].

Peptide vaccination in IDDM Similar to the use of peptide 180-188 to prevent AA [67], we found that peptide p277 can be used to treat IDDM. A single injection of pre-diabetic mice with 50 mg of p277 in oil specifically aborted the spontaneous development of IDDM [22]. Peptide therapy, similar to T cell vaccination, was associated with depression of spontaneous autoimmunity to hsp65 [22]. Indeed we find that peptide vaccination activates anti-idiotypic T cells (in preparation). Thus, regulatory networks can be entered using the epitope recognized by the autoimmune T cells as well as by using the autoimmune T cell idiotype itself. This is precisely what one would expect of a true network [11, 12, 14-16, 18].

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Immunotherapy It appears that the immune system may be able to control autoimmune responses to hsp65 and to other self antigens by antigen-specific networks of regulatory lymphocytes [11, 12, 14-16, 18, 40, 44, 48]. Regulation can be induced or enhanced by T cell vaccination, a procedure in which attenuated autoimmune T cells are used as preventive or therapeutic vaccines [2, 11, 17, 22, 28, 43]. The advantage of T cell vaccination is that it appears to work by causing depression of the disease process without noticeably interfering with the beneficial operations of the immune system. For this reason T cell vaccination is currently being tried in multiple sclerosis and in RA. A disadvantage of T cell vaccination is that each individual patient needs to have his or her vaccine tailor-made from autologous cells. One way to avoid this problem inherent in whole T cell vaccination would be to vaccinate patients with peptides derived from sequences of the TcR of the autoimmune T cells. The feasibility of this approach has been demonstrated in the EAE model in experiments using peptides from the V~8 sequence [44, 52], or from the clonally specific VDJ region of the TcR [31]. The success of TcR peptides in EAE may be attributed to the restricted use of V~ gene segments by the pathogenic T cells in the Lewis rats and PL/J mice [1, 70]. These animals use the V~8.2 gene to recognize myelin basic protein [1]; thus down-regulation of the use of V~8.2 T cells can prevent EAE [44, 52]. If any spontaneous autoimmune diseases of humans would manifest usage of restricted and predictable TcR genes, then V gene peptides might be a convenient way of obtaining the effects of T cell vaccination without the need for whole cells. Note that peptides from a domain of the TcR common to all the T cells using that particular V gene could vaccinate the individual against all T cells that happen to use that V gene for recognition of any antigen and not just for the autoimmune target antigen. Therefore, vaccination with such a peptide could incur the cost of a broader immunosuppression than is ideally required to treat the particular disease. Peptides from the VDJ region would be more specific [31]. Another factor to keep in mind is that T cell vaccination appears most effective when the T cells used for vaccination activate the natural anti-idiotypic network [12, 14-16, 18]. Thus, the most effective TcR peptides would be those recognized by the natural regulatory T cells. TcR peptide vaccination might, therefore, be optimized by defining and using the natural regulatory epitopes of the TcR. Antigen-specific regulation of autoimmune processes may also be achieved using peptides derived from the target antigen. This has been demonstrated by the use of the hsp65 peptides reviewed here [22, 66, 67] and in the use of epitopes of myelin basic protein to inhibit EAE [8, 26]. Using part of the antigen itself to actively strengthen regulatory processes, we can avoid the problems of using tailor-made T cell vaccines or of defining TcR peptides in situations where TcR gene usage is not uniform in all persons suffering from a given disease. We simply can give the proper peptide epitope of the antigen in a suitable form and let each patient organize his or her own set of regulatory cells in response to the peptide, irrespective of differences in the idiotypes or

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networks operating in the population. This, of course, assumes that a large part of the patient population will respond to a defined set of autoantigen peptides. It appears that autoimmune diseases are characterized by restricted sets of autoreactivities to dominant self antigens. In other words, the immune reactivities of patients with the same diseases are quite similar. The reasons for the dominance of particular self antigens such as hsp65 or myelin basic protein are as yet unknown. One of us (I.R. Cohen) has proposed the idea that a limited set of regulatory networks both regulates autoimmune reactivity and in fact operationally defines the dominance of certain autoantigens. This is discussed elsewhere [10, 12, 16, 18]. The point to be noted here is that the dominance of the key autoantigens such as hsp65 may make it feasible to develop and use peptide therapies based on epitopes of target autoantigens. Whether peptide therapy will be directed by the TcR or by the target autoantigen obviously will have to take into account their effectiveness in activating regulatory networks. The decision will also depend on the heterogeneity inherent in the biology of autoimmunity. Which are more restricted, the usage of TcR genes or the choice of self epitopes? A third use of peptides in immunotherapy is to block the interaction of the autoimmune T cell with the complex of target epitope-MHC-presenting molecules (see [36, 38, 64]). In this case, one designs a peptide that binds more strongly to the MHC molecule than the natural peptide that serves to activate the critical autoimmune T cells. The blocking peptide acts as a competitive inhibitor at the level of antigen presentation and the immune system is blinded. Therapy using MHC-blocking peptides assumes that autoimmune diseases are restricted to certain MHC allotypes, an assumption for which there is good evidence in certain diseases. The approach also assumes that blocking peptides can be designed that will block only the MHC molecule involved in the disease and leave free the other MHC molecules to service the needs of beneficial immune recognition. It seems, however, that different MHC molecules can bind the same peptides quite well and that a single peptide might be bound by different MHC molecules [45, 46]. Therefore, it may be difficult in practice to obtain a blocking peptide completely specific for a single MHC. The other problem of peptide blockers is that they would have to be used continuously and may induce immune or allergic reactions to themselves. It is conceivable, however, that blocking peptides might actually aid active regulatory networks by interfering, even temporarily, with the activation of virulent autoimmune T cells. Thus, blocking peptides might have long-term beneficial effects far beyond simple blocking. The history of therapy teaches that no single treatment modality solves all problems and we should expect that different forms of specific immunotherapy, or even combinations of immunotherapies, will turn out to be most suited to particular diseases or individual patients. Progress in specific immunotherapy depends ultimately on our ability to identify the specific target antigens critical to each disease. If you have the antigen, then you get the T cells and characterize their receptors, identify directly the MHC-presenting molecules, design peptides rationally, diagnose the autoimmune disease and follow its response to specific therapy.

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Discovery of the involvement of hsp65 and its various epitopes in AA and I D D M has opened up new views of the physiology of immune regulation, the pathophysiology of autoimmune disease, and the therapeutic potential of basic information. Although the questions raised by hsp65 immunity as yet far outweigh the answers provided, the prospects are most satisfying.

Conclusions hsp are molecules which are highly conserved from procaryotes to eukaryotes. At a first glance the immune system should treat these molecules as self. However, strong immune reactions to bacterial hsp are observed during infection in mammals. hsp65 plays a role in several autoimmune diseases in animal models. In AA in Lewis rats the involvement of hsp65 has been revealed by T cell clones which induce disease in naive recipients, or by T cell vaccination experiments. T cell clones which show in vivo activity have been used as tools in vitro to define epitopes involved in the disease process. In this manner mycobacterial hsp65 and its epitope peptide 180-188 were deduced for AA in Lewis rats. Similarily the epitope p277 was defined for diabetes in N O D mice. The role of hsp65 in several other autoimmune diseases was seen when animals were pretreated with hsp65 and found to be protected from subsequent induction of autoimmune disease. F r o m the involvement of hsp65 in several different autoimmune diseases, it would appear that hsp65 is somehow a key factor in natural autoimmunity. At a fist glance this is surprising since mycobacterial hsp65 shows 50% amino acid homology with human hsp65, in other words it is ' h a l f - s d f ' . Peptide epitopes, peptide 180-188 in AA in Lewis rats and p277 in I D D M in N O D mice, have been used for peptide vaccination, which represents another possibility for prevention of autoimmune disease. The immunological mechanism which leads to resistance from autoimmune disease involves hsp65 immunity and appears not to be associated with tolerance or non-responsiveness to hsp65, but seems to be due rather to modulation of naturally existing networks of idiotypeanti-idiotype T cells organized around hsp65 as the target antigen.

Acknowledgements We thank I. Wiesenberg for helpful discussions and critically reading the manuscript. Part of this work was supported by grants NS-23372 and AM-32192 from the National Institutes of Health, by the Juvenile Diabetes Foundation International, by the Merieux Foundation, by the Minerva Foundation, and by Tauro and Mr. Rowland Schaeffer. IRC is the incumbent of the Mauerberger Chair of Immunology. References 1. Acha-Orbea H, Steinman L, McDevitt HO (1989) T cell receptors in murine autoimmunediseases. Annu Rev Immunoi 7:371 2. Ben-Nun A, Wekerle H, Cohen IR (1981) Vaccination against autoimmune encephalomyelitis with T-lymphocyte line cells reactive against myelin basic protein. Nature 292:60 3. Billingham MEJ (1983) Models of arthritis and the search for anti-arthritic drugs. Pharmacol Ther 21:389 4. Billingham MEJ, Carney S, Butler R, Colston MJ (1990) A mycobacterial 65-kDa heat-shock protein induces antigen-specific suppression of adjuvant arthritis, but is not itself arthritogenic. J Exp Med 171:339

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