HLA-B27 is highly associated with ankylosing spondylitis (AS), but the mechanism is unknown. Among the HLA-B27 alleles, B*2709, which differs by one amino acid from the susceptible B*2705, is not associated with the disease. Here, we analyze the reactivity, in patients with AS and in healthy controls carrying the B*2709 or B*2705 alleles, to an EBV epitope derived from LMP2 (236-244) and to a sequence-related self-peptide from vasoactive intestinal peptide receptor 1 (VIP1R 400-408). We found that both B*2705+ and B*2709+ subjects possess LMP2 236-244–specific, HLA-B27–restricted T cells, whereas only the B*2705+ individuals respond significantly to VIP1R 400-408. These results prompted us to compare, by IFN-γ ELISPOT analysis, the T-cell response to VIP1R 400-408 in patients with AS versus B*2705 healthy controls. The data show that VIP1R 400-408–specific reactivity is a major feature of the patients with AS. These findings show, for the first time to our knowledge, a widespread reactivity in patients with AS against a self-epitope that exhibits some features of a putative “arthritogenic” peptide.
Maria T. Fiorillo, Monica Maragno, Richard Butler, Maria L. Dupuis, Rosa Sorrentino
Submitter: Robert A. Colbert, MD, PhD | bob.colbert@chmcc.org
Division of Rheumatology, Children's Hospital Medical Center, Cincinnati, OH 45229
Published September 7, 2000
The paper by Fiorillo et al. is of interest as they provide evidence that increased cytotoxic T lymphocyte (CTL) autoreactivity against HLA-B27 may occur in patients with ankylosing spondylitis (AS). They show that B27-restricted CTL recognizing a self-peptide derived from the vasoactive intestinal peptide receptor (VIP1R 400-408) can be expanded from the peripheral blood of patients with ankylosing spondylitis (AS), but not healthy individuals expressing B*2705 or B*2709 (the latter being a subtype that may not be associated with disease). To investigate the lack of responsiveness in individuals with B*2709, the authors seek to determine whether this subtype of B27 can present VIP1R 400-408. It is shown that CTL lines derived from B*2705(+) AS patients can recognize the VIP1R epitope added to T2 cells expressing B*2709, and that B*2709 can be stabilized on the surface of these cells after 18-20 hrs of continuous incubation with the peptide (T2 cell surface stabilization assay). Interestingly, B*2709 stabilization is somewhat more efficient than B*2705, leading the authors to conclude that B*2709 binds the VIP1R epitope with higher affinity than B*2705, and that lack of responsiveness in individuals with B*2709 cannot be due to impaired presentation. They argue instead that higher binding affinity leads to negative selection of potentially autoreactive CTL, and that this is the basis for lack of responsiveness and could even underlie the differential disease association.
Several studies suggest that this conclusion is premature, and may be incorrect. In an analogous situation, Brooks et al. have shown that individuals with B*2702 respond to an EBV epitope (EBNA 3B 243-253) whereas those with B*2705 do not (1). CTL lines from B*2702 individuals recognize this epitope when it is added to target cells expressing either B*2705 or B*2702, and it binds to B*2705 even better than B*2702 as judged by the T2 cell surface stabilization assay. However, B*2702 does not present the EBNA 3B epitope when it is expressed in target cells using recombinant vaccinia virus. Interestingly, there is at least a four fold difference in the stability of B*2705 and B*2702:peptide complexes, with the non-immunogenic B*2705 complexes falling apart much more rapidly than B*2702 complexes. Thus, the stability of B27:peptide complexes correlates with presentation and immunogenicity much better than binding determined using the T2 cell surface stabilization assay, which has been noted to primarily reflect peptide loading ability or on rate (1). Several other examples of a correlation between immunogenicity and the stability of MHC:peptide complexes have been published (2-5). Studies that correlate affinity with immunogenicity, are generally based on equilibrium peptide binding assays (6). Thus, inefficient presentation may indeed account for the lack of responsiveness to this epitope in B*2709 individuals.
Even if VIP1R 400-408 is not presented by B*2709 it may still fit criteria for an arthritogenic peptide. In fact, it has been more typical to rationalize that arthritogenic peptides would be presented only by alleles that are associated with disease (7), rather than the argument used by Fiorillo et al. in this paper that high affinity binding protects against arthritogenicity. This raises the question of whether the approach of establishing differential binding to B27 subtypes is rational in predicting whether a peptide might be arthritogenic. If so, should the peptide bind better or worse to the non-disease-associated allele? To complicate this issue B*2709 has now been reported in individuals with spondyloarthropathy (8), raising the possibility that genetic susceptibility or protective factors differ in the Sardinians where B*2709 is not associated with disease. There is precedent for this in west Africans where B*2705 and B*2703 do not appear to cause disease (9). Until the association or lack of association between B27 subtypes and disease can be established in multiple populations or perhaps even animal models, it would seem prudent not to let the approach to solving arthritogenicity be dictated by differences between B27 subtypes.
1. Brooks, J. M. et al. 1998. HLA-B27 subtype polymorphism and CTL epitope choice: studies with EBV peptides link immunogenicity with stability of the B27:peptide complex. J. Immunol. 161:5252-5259.
2. Van der Burg, S. H. et al. 1996. Immunogenicity of peptides bound to MHC class I molecules depends on the MHC-peptide complex stability. J. Immunol. 156:3308-3314.
3. Levitsky, V. et al. 1996. The life span of major histocompatibility complex-peptide complexes influences the efficiency of presentation and immunogenicity of two class I-restricted cytotoxic T lymphocyte epitopes in the Epstein-Barr virus nuclear antigen 4. J. Exp. Med. 183:915-926.
4. Sijts, A. J. A. M. et al. 1997. Enhanced intracellular dissociation of major histocompatibility complex class I-associated peptides: a mechanism for optimizing the spectrum of cell surface- presented cytotoxic T lymphocyte epitopes. J. Exp. Med. 185:1403-1411.
5. Levitsky, V. et al. 1997. Natural variants of the immunodominant HLA A11-restricted CTL epitope of the EBV nuclear antigen-4 are nonimmunogenic due to intracellular dissociation from MHC class I:peptide complexes. J. Immunol. 159:5383-5390.
6. Sette, A. et al. 1994. The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes. J. Immunol. 153:5586-5592.
7. Fiorillo, M. T. et al. 1997. Susceptibility to ankylosing spondylitis correlates with the C-terminal residue of peptides presented by various HLA-B27 subtypes. Eur. J. Immunol. 27:368-373.
8. Khan, M.A. 2000. HLA-B27 polymorphism and association with disease. J. Rheumatol. 27:1110-1114.
9. Brown, M.A. et al. 1997. Ankylosing spondylitis in West Africans - evidence for a non-HLA-B27 protective effect. Ann. Rheum. Dis. 56:68-70.
Submitter: Maria Teresa Fiorillo and Rosa Sorrentino | Sorrentino@axcasp.caspur.it
Department of Cell Biology and Development, University of Rome "La Sapienza"
Published August 23, 2000
We apologize for not having quoted the primary references by Geczy and colleagues, although we were aware of their pioneering work. However, we judged the review by Benjamin and Parham (1) as the most suitable place where to learn of this as well as other relevant findings appeared in the literature before 1990. This especially considering the long-lasting debate, extensively reported by B. and P., that followed the description of the Geczy factor and whose comprehensive account was beyond the purpose of our communication. The later paper by Hermann and colleagues (2) was dutifully quoted in the article.
In acknowledging the work previously done by Geczy and colleagues as well as by others, we take the opportunity to stress that, to the best of our knowledge, the anti-VIP1R reactivity described by us, while apparently not stemming from a cross-reaction with bacterial proteins, represents the first sequence-specific, anti-self reactivity shown to be present in a number of patients with AS at considerable higher proportion than in controls. In this respect, it represents an undeniable support to the defenders of the arthritogenic, self-peptide/s hypothesis. However, the understanding of the connection, if any, between this autoreactivity and bacterial infections as well as between this or any other CD8+ T cell reactivity and disease pathogenesis, entails different approaches and a welcome collaborative effort.
Sincerely,
Maria Teresa Fiorillo and Rosa Sorrentino Dept. of Cell Biology and Development University "La Sapienza" Via degli Apuli, 1 00185 Roma (Italy)
1. Benjamin, R., and Parham, P. 1990. Guilty by association: HLA-B27 and Ankylosing Spondylitis. Immunol. Today. 11:137-142.
2. Hermann, E., Yu, D.T.Y., Meyer zum Buschenfelde, K.H., and Fleisher, B. 1993. HLA-B27-restricted T cells derived from synovial fluids of patients with reactive arthritis and Ankylosing Spondylitis. Lancet. 342:646-650.
Submitter: Andrew F.Geczy and John S.Sullivan | ageczy@arcbs.redcross.org.au
ARCBS-NSW/ACT;153 Clarence St SYDNEY,2000.AUSTRALIA
Published August 23, 2000
We read with interest the article by Fiorillo et al. which claims to present for the first time evidence that an "arthritogenic" peptide in association with HLA-B27 might be involved in the pathogenesis of ankylosing spondylitis (AS). We wish to draw the authors' attention to our previous work (1), which clearly showed that cytotoxic T lymphocytes (CTLs) could be raised by stimulating the peripheral blood mononuclear cells (PBMCs) of an HLA-B27-positive clinically normal individual with the PBMC of an HLA-identical sibling suffering from AS; such CTL could specifically lyse PBMC from B27-positive AS patients but not PBMC B27-positive or- negative normal individuals. Moreover, these disease-specific CTL will lyse B27-positive PBMC that have been modified in vitrowith culture filtrate from one of our "arthritogenic" bacteria (2). Qualified support for the concept of a "disease-associated"CTL has been provided by the studies of Hermann and her associates (3), who showed that bacteria such as Yersinia and Salmonelle can generate B27-restricted bacteria-specific CTL. It would be helpful if the authors had cited these primary references, especially those that preceded the article by Benjamin and Parham. In particular, the authors should be aware of the blind confirmation of our work by van Rood's group (4) some 15 years ago and in a further double-blind trial in the same year (5).
1. Geczy, A.F., McGuigan, L.E.,Sullivan, J.S., and Edmonds, J.P. 1986. Cytotoxic T lymphocytes against disease-associated determinant(s) in ankylosing spondylitis. J. Exp. Med. 164:932-937.
2. Geczy, A.F., and Sullivan, J.S. 1995.Possible role of HLA-B27 associated cytotoxic T lymphocyte activity in the pathogenesis of the seronegative arthropathies. Ann. Rheum. Dis. 54:329-330.
3. Hermann, E., Yu, D.T.Y., Meyer zum Buschenfelde, K.H., and Fleischer, B. 1993.HLA-B27-restricted T cells derived from synovial fluids of patients with reactive arthritis and ankylosing spondylitis. Lancet. 342:646-650.
4. van Rood, J.J., et al. 1985. Blind confirmation of Geczy factor in ankylosing spondylitis. Lancet. ii:943-944.
5. Geczy, A.F., van Leeuwen, A., van Rood, J.J., Ivanyi, P., Breur, B., and Cats, A. 1985. Blind confirmation in Leiden of Geczy Factor on the cells of Dutch patients with ankylosing spondylitis. Hum. Immunol. 17:239-245.