Journal of Autoimmunity (1992) 5 (Supplement A), 205-208

Towards Peptide Immunotherapy in Rheumatoid Arthritis: Competitor-Modulator Concept

M a r c a H. M. W a u b e n , C l a i r e J. P. B o o g , R u u r d v a n der Zee a n d Willem van Eden Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, University of Utrecht, P.O. Box 80.165, 3508 TD Utrecht, The Netherlands

By the introduction of single-amino acid substitutions in well-defined T cell epitopes of a u t o i m m u n o g e n i c proteins, e.g., mycobacterial heat shock protein (hsp60) in adjuvant arthritis (AA) and myelin basic protein (MBP) in experimental allergic encephalomyelitis (EAE), efficiently blocking MHC binding peptides were selected. Despite the finding that a substituted variant of epitope 180--188 of hsp60 was 'blocking' not only responses of the 180-188 specific arthritogenic T cell A2b, but also responses of the M B P specific encephalitogenic T cell Zla, in vivo testing of this c o m p e t i t o r peptide revealed a very p r o m i n e n t disease inhibitory activity in AA but not in M B P - i n d u c e d EAE. The selectivity of this peptide in suppressing the disease in which native 180-188 appears to be of critical relevance, offers the possibility of achieving disease specific i m m u n o l o g i c a l intervention. Based on the results collected so far, it seems that, in vivo in addition to blocking activity, a variant peptide itself could trigger responses that confer protective activity in AA. Such c o m b i n e d activities m a y well be r e q u i r e d for achieving full in vivo inhibition of a disease in which multiple distinct epitopes m a y play a role, possibly t h r o u g h presentation by m o r e than one MHC product.

Specific immunological intervention in autoimmune diseases seems largely dependent on the proper definition of antigens involved. In contrast to experimentally induced autoimmunity where a relevant antigen is deliberately selected, in h u m a n disorders information concerning antigens as determinants of the autoimmune process is frequently lacking. However, with the advent of new techniques such as T cell cloning from sites of inflammation, progress is being made. In this respect, attention is currently focused on heat-shock proteins (hsp) in the initiation or perpetuation of the arthritic inflammatory process [1]. Both with regard to the h u m a n and the (myco)bacterial hsp60, enhanced immunity, at the level of B and T cells, has been claimed to coincide with the expression of arthritic diseases [2-5]. 205 0896-8411/92/0A0205+ 04 $03.00/0

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Furthermore, raised levels of expression of hsp60 in the inflamed synovial tissue has been seen to occur [6, 7]. Where disease is induced in experimental models of arthritis either by bacterial antigens (adjuvant arthritis, streptococcal cell-wall induced arthritis) or other reagents (collagen arthritis, pristane arthritis etc.), T cell responses to mycobacterial hsp60 have been found to develop along with arthritis. Furthermore, in these models, preimmunization with mycobacterial hsp60 has been shown to protect against the induction of disease [1]. These findings suggested that T cells that recognize hsp60 were taking part in the autoimmune regulatory responses associated with synovial inflammatory processes. T h e initial observations concerning the potential relevance of hsp60 were made in the model of mycobacteria induced arthritis, adjuvant arthritis (AA). T cell clone A2b, isolated from a Lewis rat with AA, was found to be capable of transferring AA into naive irradiated animals [8]. Furthermore, a related subclone, A2c, and attenuated A2b itself, were found to confer protection against AA [9]. Subsequent findings that A2b and A2c were specific for the 180-188 sequence of mycobacterial hsp60, have indicated the critical contribution of hsp60 and the 180-188 sequence in particular to the process of AA [ 10]. T cell responses with specificity for epitope 180-188 of hsp60 can be seen in Lewis rats after immunization with Mycobacterium tuberculosis (Mr). When rats are immunized intracutaneously at the base of the tail to induce arthritis (day 0), responses against 180-188 may be observed preferentially at the site of draining inguinal lymph nodes around day 14. This is around the time that the macroscopic signs of arthritis begin to manifest themselves. In contrast to this, in Fisher rats no responses to epitope 180-188 have been observed following immunization with Mt, despite the fact that, at least for M H C Class II products, Lewis and Fisher are identical [11]. In terms of arthritis susceptibility, however, Fisher and Lewis rats are different: Fisher rats are notoriously resistant to AA whereas Lewis rats are very susceptible. T h u s , although Fisher rats are equipped with the proper M H C products to select epitope 180-188 for T cell recognition, this strain is a nonresponder to 180-188. Also priming with peptide 180-188 itself in IFA, was found to be successful in Lewis and not in Fisher rats. However, interestingly, in exceptional cases of arthritis developing in Fisher rats, responses to 180-188 were obtained [ 11 ]. T h e latter findings indicated the association between arthritis development and 180-188 responsiveness, and suggest the importance of such responses for the development of disease, at least in context of the Lewis and Fisher M H C haplotypes. Further indications of the critical significance of 180-188 for arthritis development, were the observations that preimmunization with 180-188 led to resistance against subsequent induction of AA by M t immunization [12]. Thus, responses to 180-188 were seen to occur not only in association with AA development but also seemed to lead to protection, which was likely to develop in a similar way to the protection seen as a result of overt AA. It is a well-known phenomenon that following AA, Lewis rats develop resistance to subsequent attempts of inducing AA. However, administration of peptide 180-188 at the time of AA induction has not been shown to influence AA severity. In the course of experiments designed to investigate the relative contribution of individual residues within the 180-188 sequence for recognition by the arthritogenic T cell clone A2b, we have tested multiple variant peptides for inducing A2b responsiveness or for competition with the native 180-188 peptide for M H C binding

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using A2b proliferation as a functional read-out system. Whereas the n u m b e r of substituted peptides that induced A2b to respond appeared to be relatively rare, it was relatively easy to generate M H C binding competitors. U p o n testing the most powerful competitor peptide for inhibiting responses of another T cell line, the encephalitogenic line Z l a , strong competition was seen. Both A2b and Z l a were known to use the I-A equivalent of the Lewis rat M H C (RT.1B 1) as a restriction element. Since our 180-188 epitope was presented to T cells via the B locus product, different T cells, restricted by RT-1 B, were sensitive to inhibition by a nonstimulatory variant of the native B locus product binding peptide. Since Lewis rats are susceptible not only to AA but also to myelin basic protein-(MBP) induced EAE, we had an opportunity to test, for their inhibitory activities in vivo in the respective disease models, the B locus product binding peptides, which were so efficiently blocking the in vitro responses of the disease inducing T cells. Unexpectedly, it appeared that the addition of the non-stimulatory substituted variant of 180-188 to the inoculum used for disease induction (whole mycobacteria in oil for AA, M B P in CFA for EAE) inhibited the development of AA and not of EAE. Thus, despite the fact that the 180-188 related variant inhibited, indiscriminately, in vitro arthritogenic and encephalitogenic effector T cells, the in vivo disease inhibitory activity turned out to be disease specific. This suggested that MHC-blocking by itself was not sufficient to inhibit diseases induced by multivalent antigens such as whole mycobacteria or MBP. In support of such a conclusion were recent findings that other nonstimulatory peptides with strong in vitro blocking activities, designed as substitution variants of the M B P sequence recognized by T cell Z la, were not capable of inhibiting the induction of AA. Therefore, we had to conclude that the blocking variant of 180-188 was interfering with induction of AA by qualities that were beyond the capacity of binding to R T . lB. When we investigated the animals that featured inhibition of AA development following immunization with M t in the presence of the blocking peptide for in vitro non-responsiveness to the relevant antigens, we observed the following. Polyclonal T cell responses to M t were present and did not differ from control animals that had developed disease in the presence of control non-blocking peptides. However, unexpectedly, responses to the critical 180-188 sequence were raised, and certainly not inhibited, in AA protected animals compared with the controls. Furthermore, responses with specificity for the variant blocking peptide itself were observed. This indicated not only that the blocking peptide was immunogenic and had primed T cells with unique specificity for this blocking peptide, but in addition somehow the blocking variant had stimulated responses to the critical native peptide 180-188. Subsequent experiments revealed that immunization with the blocking peptide could lead to stimulation of T cells with a double specificity for both the variant and native peptide. This indicated that such T cells were related to the arthritogenic T cell clone A2b, but not identical to A2b. Since responses directed at epitope 180-188 have already been shown to be critical to both development of AA and protection against AA, it is attractive to assume that T cells triggered by the blocking peptide and cross-reactive with the native peptide 180-188 are involved in the mechanisms that may lead to inhibition of disease induction. T h e effects of preimmunization with the blocking peptide is currently being studied. However, on the basis of findings of the immunogenicity of this peptide and the specificity of the resulting

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T cell r e s p o n s e , one w o u l d h a v e g o o d r e a s o n to e x p e c t p r o t e c t i o n as a r e s u l t o f p r e i m m u n i z a t i o n w i t h t h e s u b s t i t u t e d v a r i a n t p e p t i d e . B a s e d o n t h e r e s u l t s c o l l e c t e d so far, it is c o n c l u d e d t h a t M H C c o m p e t i t i o n a n d a n t i g e n specific i m m u n o m o d u l a t i o n , i n t e g r a t e d into o n e single c o m p e t i t o r - m o d u l a t o r p e p t i d e , m a y create a p o w e r f u l tool for disease specific t h e r a p y .

Acknowledgement P a r t o f t h e w o r k was s u p p o r t e d b y a g r a n t o f P a s t e u r - M e r i e u x , L y o n , F r a n c e .

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