Unraveling the mystery of HLA-B27 association with human spondyloarthropathies using transgenic and knock out mice

seminars in I M M U N OL OG Y, Vol 10, 1998: pp 15]23

Unraveling the mystery of HLA-B27 association with human spondyloarthropathies using transgenic and knock out mice Sanjay D. Khare U , Harvinder S. Luthra† and Chella S. David U genic animals expressing this MHC class I gene.4 ] 7 The role of unknown environmental antigens in triggering the disease has also been confirmed in HLAB27 transgenic rats and mice developing spontaneous inflammatory disease.5,6,8 We have recently described a transgenic mouse model of human disease with inflammatory arthritis and nail disease as seen in Reiter’s syndrome.5,6 By breeding transgenic mice and knock out mice together, we are investigating the role of HLA-B27 in a mouse model of human disease.

Human spondyloarthropathies have a strong association with the presence of MHC class I allele, HLA-B27. Spondyloarthropathies occur predominantly in males and are usually triggered by an infection with an enterobacteria. Similar to human disease, experimental animals with HLA-B27 transgene also develop spontaneous inflammatory disease. In addition to HLA-B27, the role of environmental antigens has also been implicated in the animal models. How bacteria interact with HLA-B27 is not yet clearly understood. By breeding HLA-B27 transgenic mice with various transgenic and knock out mice, we investigated the immune mechanism in this inflammatory disease. In this review, we will summarize our recent findings and propose a hypothesis.

Unique features of HLA-B27 molecule

Key words: HLA-B27 r spondyloarthropathies r transgenic mice r animal model

Why is HLA-B27 as a class I molecule linked to an autoimmune disease? HLA-B27 molecule has a conformation similar to other MHC class I molecules with a 1 a 2 peptide binding domain and an immunoglobulin-like folding for the a 3 domain.9 Class I heavy chains Ž45 kD. bind to a 12-kD non-polymorphic b 2-microglobulin Ž b 2 m. primarily through the a 3 domain although a few residues in the a 1 a 2 domains also interact with it. The differences between B27 and other class I molecules such as HLA-A2 or -A68 is a shift in conformational loop providing relatively flexible association of a 3 with a 1 a 2 and minor packing rearrangements of a 3 and b 2 m.10,11 An additional a 3] a 1 a 2 contact is found in the HLAB27 molecule, involving a hydrogen bond from the HLA-B locus-specific residue R-239 Ž a 3. to the main chain carbonyl oxygen of R-48 Ž a 1.. In addition, incomplete covering of b sheet by a 3 and b 2 m leaves the molecule closed from both ends. It has also been hypothesized that such a concave surface might have a role in the formation of tetrameric class I molecules on the cell surface.12 Studies on crystal structure have clearly demonstrated that B and F pockets of HLA-B27 molecule are deeper and allow binding of P2, P3, P9 and to some extent P7 side chains of a nanomer peptide.11

Q1998 Academic Press Ltd

MHC association with human spondyloarthropathies THE STRONGEST ASSOCIATION of MHC genes with a human disease is between HLA-B27 and spondyloarthropathies such as ankylosing spondylitis ŽAS., reactive arthritis ŽReA. and Reiter’s disease.1,2 Although such linkage was first discovered in 1973 and has been confirmed worldwide in different ethnic groups, its role in human disease is still a mystery. Many of HLA-B27 associated diseases have an onset after an infection with certain enterobacteria.3 How these bacteria interact with HLA-B27 is still unclear. In order to understand the role of HLA-B27 in disease pathogenesis, several groups generated transFrom the U Departments of Immunology and †Rheumatology, Mayo Clinic and Medical School, Rochester, MN 55905, USA Q1998 Academic Press Ltd 1044-5323r 98r 010015q 09 $25.00r 0r si970101

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Several peptide binding studies have confirmed that arginine at P2 is a primary anchor while R, F, A or G could be secondary anchors for a B27 binding peptide.12 ] 15 Peptides longer than nine amino acids from the carboxyl terminal have also been eluted.16 Furthermore, binding of exogenously added longer peptides from the ]COOH terminal and not from the NH 2-terminal has been observed with B27 molecules in Tap deficient T2 and RMA-s cell lines ŽTable 1.. These longer peptides also follow the rule of arginine at P2 for binding. Recently, a study by Bjorkman’s group showed that peptides with substitution for arginine at P2 with methionine and glutamine can also bind to the B2702 subtype of HLA-B27 with slightly reduced affinity.17 There are 11 subtypes of the B27 allele differing from each other at few amino acids within the peptide binding groove. While most of them are linked to disease, BU 2705 and BU 2702 are highly associated and the BU 2709 subtype is not linked to spondyloarthropathies.18,19 The difference between the peptide specificity between these subtypes is that BU 2709 molecules only accept C-terminal hydrophobic residues.20 Recently, an increased prevalence of HLA-B39 antigen has been found in HLA-B27 negative patients with spondyloarthropathies in Japan.21 Since HLA-B39 and HLA-B27 have a similar B pocket, these authors suggested a role for arthritogenic peptides with common features. Stable ‘empty’ HLA-B27 molecules on viable human cells were first described by Parham’s group.22 In this study, a small number of other class I molecules such as HLA-Aw68 were also found as empty molecules but they are short lived and may not have

biological significance. These authors speculated that longlived empty B27 molecule may bind extracellular arthritogenic peptides.

HLA-B27 transgenic animals and disease To investigate the role of HLA-B27, several groups generated transgenic mice and rats in the late 1980s. At least three groups including ours reported inflammatory disease in the transgenic animals. Hammer et al 4 observed inflammatory disease with arthritis in rats with a high copy number of transgenes, HLA-B27 and human b 2 m.4 We have reported inflammatory arthritis and nail disease in transgenic mice expressing HLA-B27 or B27rhuman b 2 m in the absence of endogenous b 2 m.5,6 These mice develop disease once they are transferred from barrier facility to the conventional area. Moreover, similar to human spondyloarthropathies the disease was found to be more common in male animals compared to females. The disease begins with discoloration of nails with hyperkeratosis which lead to nail loss and inflammatory arthritis. A few male animals also developed severe ankylosis in the rear paws. Ankylosis was never observed in female animals. Histological examination showed the presence of mononuclear cells within the joint. As mice grow older, other symptoms such as splenomegaly, hair loss and skin inflammation was also observed. Similar to these studies, the role of unknown environmental antigens has also been described in transgenic rats.8 However, disease triggering agents in these animals have not yet been discovered. Transgenic animals

Table 1. Binding of longer CII-derived peptides to B27 molecules Peptides Nil NP1 Žqve control. CII. 74]85 CII. 74]88 CII. 74]90 CII. 74]92 CII. 74]93 CII. 73]92

Sequence

SRYWAIRTRSGG ARGFPGTPGLPG ARGFPGTPGLPGVKG ARGFPGTPGLPGVKGHR ARGFPGTPGLPGVKGHRGY ARGFPGTPGLPGVKGHRGYP GARGFPGTPGLPGVKGHRGY

Relative fluor. intensity 1.0 1.37 1.35 1.36 1.34 1.30 1.38 1.03

The experiment was carried out by adding 10 m M CII peptide to transporter deficient RMA-s cells co-transfected with HLA-B27 and human b 2 m at 268C. Cells were stained with B27-specific ME-1 monoclonal antibody and analyzed using a FACS-scan flow cytometer. An increase in fluorescence intensity determines stabilization of B27 molecules. A relative fluorescence intensity is shown.

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Enigma of HLA-B27 association with human diseases

used in these studies were generated using the BU 2705 subtype of HLA-B27. Another group from the Netherlands reported BU 2702 as a risk factor associated with ankylosing enthesopathy ŽANKENT. in transgenic mice.7 These investigators described ANKENT as a naturally occurring joint disease with progressive entheses in the ankle and tarsel joints of the hind paws. Although there are few differences in these experimental animal models of human disease, certain common features, such as inflammatory process in the hind paws primarily affecting male animals and role of unknown environmental factors have been confirmed. Studies from our laboratory and those of Hammer et al 4 also describe nail disease as seen in human Reiter’s disease.

Its biological significance in human disease needs to be confirmed.

Transgenic mice with peptide binding domain of HLA-B27 develop spontaneous disease We also confirmed our findings in another transgenic line ŽDB27q b 2 m8. with a 1 a 2 domain of B27 molecule and a 3 domain of mouse class I molecule, H-2K d . Transgenic mice expressing disease unrelated MHC class I genes Že.g. HLA-Cw3, H2-D d . bred into b 2 m knock out background do not develop inflammatory disease. Similar to transgenic mice with intact B27 gene, approximately 50% of DB27qb 2 m8 mice Žprimarily males. also developed nail disease and arthritis within 2 weeks after transferring them into the conventional area ŽKhare and David, manuscript in preperation.. These studies confirm that peptide binding domains of HLA-B27 contribute to the disease pathogenesis. Development of disease in DB27qb 2 m8 mice also suggests that at least one ŽQRADPHTYL. of the two B27-derived peptides suggested by Scofield and Harley 24 are not involved in the disease pathogenesis since they are derived from the a 2 a 3 domain of the B27 molecule. The other peptide, LRRYLENGK might have some role in disease pathogenesis as a similar peptide ŽRRYLENGKETL. has also been found endogenously bound to B27 molecules by other investigators.26,27

Is HLA-B27 an autoantigen or antigen presenting molecule? The exact role of HLA-B27 in disease pathogenesis is not yet clearly understood. In the early and mid1980s, a few investigators described cross-reactivity of anti-B27 antibodies with certain enterobacteria Ž Klebsiella.. Similarly, some anti-Kleb antibodies also recognized HLA-B27. 23 These experiments led these investigators to postulate that an antigenic trigger by mimicking bacteria may break tolerance against self HLA-B27 molecule in patients with spondyloarthropathies. The presence of such autoantibodies in a significant number of patients was not carefully examined. More careful studies by Scofield et al 24 showed that B27-derived peptides, LRRYLENGK which has B27 binding motif also share a sequence homology with bacterial antigens.24 They also confirmed binding of these peptides to HLA-B27 in an in vitro assembly assay.25 These investigators suggested that an antigenic trigger may break the immune tolerance against the self B27 molecule to initiate the disease process in patients. Recently, peptide elution studies by two independent groups showed the presence of a B27-specific sequence, RRYLENGKETL.26,27 This peptide sequence is slightly different than the previously described B27 sequence by Scofield et al 24 which was found naturally bound to the B27 molecule and had homology with bacterial peptides. These results confirm a hypothesis that B27-derived peptides mimicking a peptide from disease implicated enterobacteria can break the self tolerance as previously described by Scofield et al.24

b 2 m free heavy chains of HLA-B27 in disease pathogenesis We have recently reported a role for heavy chains of HLA-B27 in the disease pathogenesis.6 Unlike single transgenic mice expressing HLA-B27 with mouse b 2 m, B27rhuman b 2 m double transgenic mice lacking endogenous b 2 m develop spontaneous inflammatory disease. These two mice have significantly different levels of b 2 m free heavy chains of HLA-B27 on the cell surface. B27rhuman b 2 m double transgenic mice express a higher level of b 2 m free heavy chains of HLA-B27 on the cell surface. This study suggests that interaction of certain amino acid residues of b 2 m with HC of B27 may be important in its dissociation from the trimeric complex. In vivo treatment of these mice with a heavy chain-specific mAb ŽHC10. leads to a delay in disease development. Such an effect was not seen with an antibody against 17

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the wHC q b 2 mx complex. These experiments suggested a role for b 2 m free HC of B27 in the disease pathogenesis. Recently, b 2 m free HC of H2-L d molecules have been shown to have an altered class I binding motif.28 At this time we do not know the characteristics of peptides which may bind to b 2 m free HC of B27. Similar to H2-L d molecules, HC of B27 molecules may use a different motif to bind antigenic peptide. More investigation in this area is needed. Newly synthesized MHC class I molecules bind to b 2 m and peptides in the endoplasmic reticulum and the trimeric complex is transported to the surface of the cell.29 Strong interaction of b 2 m and heavy chain is critical for a stable complex. Low affinity binding of b 2 m and peptides with B27 HC may result in dissociation of b 2 m from the complex.30 In such a situation, endogenously bound peptides could ‘pop out’ leading to b 2 m and peptide-free HC. Depending on the half-life of these molecules, they may bind extracellular peptides as previously suggested by others.31,32 Thus b 2 m free heavy chains can contribute to the disease susceptibility by: Ž1. presenting exogenous antigens to T cells; or Ž2. become an autoantigen presented by self MHC molecules.

a 1 a 2 domains, while 8, 10, 11, 12, 24, 63, 70 and 99 interact with the a 3 domain of MHC class I molecule. Considering these studies, additional amino acid residues at position 34 ŽHrD., 51 ŽMrH. and 54 ŽMrL. of b 2 m may also be critical in their interactions with the HLA-B27 molecule. Similarly, mutation in b 2 m have also been shown to affect its interaction with HLA-B27 molecule.33 Such interaction leads to low cell surface expression, altered affinity of wHC q mutant b 2 mx with peptide and CTL recognition. Polymorphism in a few amino acid residues of the MHC class I molecules affects stability of association with b 2 m and also peptide binding. Studies with murine class I molecules ŽH-2D d , H-2K b versus H2Ld , H-2D b . showed that glutamic acid at position 9 contribute to the stability of complex.34 The E9V mutation at this position resulted in a more stable complex, while it lost peptide presentation to specific T cell hybridoma. At position 9, histidine in B27 instead of tyrosine in most other HLA-B molecules could also be important for its interaction with b 2 m.

Is there any role for MHC class II molecules in disease pathogenesis? Being an MHC class I mediated disease, CD8 q T cells recognizing an antigenic peptide with an affinity for the HLA-B27 molecule has been suggested.35 Effector CD8 mediated T cell response is, however, a delayed event and is somehow dependent on the activation of CD4 positive T-helper cells recognizing a longer peptide in the context of MHC class II molecules.36 One can postulate that disease pathogenesis is triggered by the presentation of peptides by certain class II molecules to CD4 q T cells and providing initial help for effector CD8 function. An increased association of HLA-DRB1U 0408 andDQB1U 0301 in HLA-B27 linked reactive arthritis have been shown in some studies.37,38 The exact role of these molecules and their association with B27-linked diseases need to be investigated. On the other hand, presentation of B27-derived peptides by the MHC class II molecules has recently been suggested by several investigators.18,39,40 In order to investigate such a scenario, we bred H2-A knock out gene ŽA b 8. 41 into the HLA-B27 transgenic mice ŽB27 q b 2 m8A b 8..42 These mice do not express endogenous MHC class II molecules, H2-A and H2-E. When transferred from the barrier facility to the conventional area, B27 q b 2 m8A b 8 mice developed nail disease and arthritis as previously described in

b 2 m and its interaction with HLA molecules Interaction of non-polymorphic b 2 m molecule with MHC class I molecule is important in the peptide binding. Studies on the crystal structure of HLA-A2 molecule showed that at least six, five and nine amino acid residues of b 2 m molecule interact with the a 1, a 2 and a 3 domain of MHC class I molecules, respectively.10 Crystallographic studies on HLA-B27 also showed similar patterns.11 Our studies showed that transgenic mice expressing B27rhuman b 2 m develop spontaneous disease while mice expressing B27rmouse b 2 m do not. This led us to postulate that some amino acid residues of b 2 m interacting with MHC molecule may play a role in the disease. While aligning the sequence of B27 with published data of the crystal structure of HLA-A2,10 we observed that valine at position 12 on the a 1 domain of HLA-B27 interacts with serine of human b 2 m instead of proline of mouse b 2 m at position 33. Differences in mouse and human b 2 m at position 22 and 97 and their interaction with a 3 domain may also be important. In a recent review, Joyce and Nathenson28 summarized that amino acid residues at positions 1, 31, 34, 51, 53, 54 and 60 have closer proximity with 18

Enigma of HLA-B27 association with human diseases

B27 q b 2 m8 mice.42 These results show that MHC class II molecules do not have a role in the disease pathogenesis at least in the mouse model.

ence in the binding of small or longer peptides to the HLA-B27 molecule ŽTable 1.. Biological significance of longer peptides bound to B27 molecules have not yet been determined.

Endogenous versus exogenous antigens in disease pathogenesis

CD4 versus CD8 T cells in the pathogenesis of B27-linked disease

Many B27-associated spondyloarthropathies begin after an infection with Gram negative enterobacteria such as, K. pneumoniae, S. flexneri, S. typhemurium, etc. Although processing and presentation of such exogenous antigens is usually mediated through MHC class II molecules, presentation of these antigens by class I molecules including HLA-B27 has also been shown in some experimental conditions.43,47 In the classical class I presenting pathway, proteosome complex ŽLmp-2, Lmp-7 etc. process the antigens and small antigenic peptides are translocated through Tap1rTap2 heterodimers to the endoplasmic reticulum for assembly.48 The role of polymorphism of proteosome and transporter genes in the human disease has been controversial. Increased frequency of Tap1C and Tap2A has been shown to be associated in patients with B27-linked Reiter’s disease.49 Tap 1B was particularly found more frequently in patients with ankylosing spondylitis.50 Higher prevalence of certain Lmp genes in B27-associated diseases has also been reported.51 ] 54 None of these studies describe influence of such Tap and Lmp polymorphism in the peptide selection by HLA-B27 molecule. The effect of Tap polymorphism on peptide selection was first described in HLA-B27 transgenic rats with polymorphic Tap alleles without affecting disease susceptibility.55 Similarly, we also did not find any effect of Tap and Lmp polymorphism in our transgenic mouse model ŽKhare and David, unpublished data.. In order to directly investigate whether Tap genes have some role in the B27-associated diseases, we bred Tap1 knock out gene ŽTap18. 56 into B27 transgenic mice. B27qb 2 m8 mice with or without human b 2 m transgene and lacking Tap1 gene developed spontaneous disease suggesting that processing of antigenic peptide in this model does not require the transporter genes.57 Processing of exogenous antigens and Tap independent transport of these peptides need to be investigated. A previous study has shown that peptides with a molecular weight from 900 to 4000 Žeight to 33 amino acid residues. bind a subpopulation of MARB4 reactive B27 molecules. In vivo delivery of longer peptides require a peptide transporter. In our studies, we did not find a differ-

The role of CD4 versus CD8 q T cells in B27-linked disease has been controversial. Although as a MHC class I associated disease it should be mediated by CD8 q T cells, many studies have reported more CD4 q T cells compared to CD8 q T cells in the affected tissues in patients with reactive arthritis. Moreover, some of these CD4 q T cells were found to be restricted by MHC class II molecules. Do they provide help to the B27 restricted CD8 effector function? Only a few studies have described B27 restricted bacterial antigen-specific CD8 q T cells from patients with AS and ReA. To determine the contribution of CD4 cells in the disease pathogenesis, we bred the mutated CD4 gene 58 into B27 transgenic mice. HLAB27 transgenic mice lacking CD4 q T cells ŽB27q b 2 m8CD48. showed a significantly reduced incidence of nail disease and arthritis ŽKhalil et al, unpublished data.. This study suggests that CD4 q T cells may play a critical role in the onset of inflammation and arthritis in the mouse model. Whether pathogenic CD4 q T cells are HLA-B27 restricted or merely critical for providing the help to the CD8 effector function needs to be explored. Experiments involving cell transfer from a wild type diseased animal to disease resistant CD4 null mice are in progress to confirm these findings. Further, we are also breeding CD8 null gene 59 in B27 transgenic mice to investigate a direct role of CD8 q T cells.

Possible triggering antigens and target tissue On the basis of various tissues which are affected in the B27 linked diseases, type II, type IX, type XI collagen as well as proteoglycans may be candidates as targets for an autoimmune attack in transgenic animals and patients with spondyloarthropathies. Moreover, types II and XI collagen are common denominators while comparing affected target tissues with its presence in the eyes and joints in B27-linked acute anterior uveitits and several arthritidis. A study by Guo et al 60 described collagen peptide-specific T cells in a few AS patients. Type II collagen is a large 19

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Table 2. Collagen ŽCII, CIX and CXI.-derived peptides with B27 binding motif Collagen sequence

Peptides with B27 binding motifs

CIX.71]79 CIX.248]256 CXI.1555]1563

1. RRXXXXXXX RRAIQRVVG RRETCHELP RRHTEGMQAU 2. XRXXXXXXH LRPRRETCH 3. XRXXXXXXXL GRDGEPGTL PREKGPDPL TRNLYPSGL YRVSKQAQL QRKNSEDTL 4. XRXXXXXXXA GRTGPAGAA MRVIQEHFA RRHTEGMQAU 5. XRXXXXXXXF IRAEGNSRF TRILDEEVF 6. XRXXXXXXXG ARGNDGQPG ERGAPGNRG GRGIPGPPG QRGPPGEAP ARGPTGKPG LRGFPGERG 7. XRXXXXXXXR LRCQGQDVR GRPGADGGR 8. XRXXXXXXXK GRDGAAGVK NRIESLPIK FRYGGDGSK

CIX.245]253 CII.81]89 CII.1159]1167 CIX.99]107 CXI.74]82 CXI.351]359 CII.264]272 CII1122]1130 CIX.767]775 CXI.1555]1563 CII.1352]1360 CXI.208]216 CII.274]282 CII.439]447 CIX.356]364 CIX.506]514 CXI.893]901 CXI.1025]1033 CII.21]29 CXI.580]588 CII.978]986 CIX.199]207 CXI.497]505

several homologous sequences with enterobacteria such as Klebsiella, Salmonella, Shigella and Yersinia which have been suggested as triggering agents in B27-associated spondyloarthropathies ŽTable 3..

HLA-B27 and spondyloarthropathies: a hypothesis In the thymic school of T cell education, immature lymphocytes are taught not to react against self which is primarily based on affinityravidity of peptides with MHC molecules. High affinity interaction of T cells with MHCrpeptide complex lead to death or anergic state Žnegative selection., while cells with intermediate and low affinity interaction lead to positive selection which would go to the periphery. Thus, it is possible that a few autoreactive T cells may escape the negative selection and reach the periphery. If these cells do not see any antigen in their life, they will die. If these T cells encounter a mimicking environmental antigen, they will expand in HLA-B27 associated spondyloarthropathies. It is possible that T cells reacting with a peptide from cartilage are escaping negative selection in the thymus. How peptides interact with b 2 m associated and b 2 m free B27 molecules in the thymus may also have an implication for T cell selection. In this situation, a peptide interacting with HC q b 2 m may delete the T cells, however, autoreactive T cells interacting with the same peptide in the context of b 2 m free heavy chains could escape clonal deletion in the thymus. Several collagen peptides with the B27 binding motif share a sequence homology with peptides from enterobacteria implicated in human spondyloarthropathies ŽTable 3.. Presentation of these peptides by B27 in the periphery could expand these autoreactive T cells before they die in the periphery. Considering the tissue specificity, these cells will then migrate to joints or the eyes, proliferate and cause tissue damage ŽFigure 1..

An example of collagen-derived peptides with HLA-B27 binding motif. The search was conducted by analyzing XRXXXXXXŽRrKrHrLrFrG. motif for B27 binding on sequences of collagen molecule. Sequence patterns were analyzed by ‘findpattern’ using the GCG sequence analysis program. A few examples are shown.

molecule Ž) 1100 amino acids. and have several repeats of arginine and glycine. By using a recent motif register for B27 binding, we found 60, 36 and 49 peptides on type II, IX and XI collagen, respectively, with the appropriate motives for B27 ŽTable 2.. Proteoglycan is more than 2400 amino acids long and has 41 peptides which may bind to the HLA-B27 molecule. B27 transgenic mice lacking b 2 m, immunized with type II collagen inside the pathogen-free colony developed arthritis suggesting CII could be one of the major targets in the B27-linked diseases ŽLee et al, unpublished data.. Type II collagen has

A final note The exact role of HLA-B27 in the pathogenesis of human disease has not yet been determined. In the mouse model of B27-linked human spondyloarthropathies, the HC of the B27 molecule seems to be critical in disease pathogenesis. The heavy chain of B27 molecules are also expressed in humans. Their 20

Enigma of HLA-B27 association with human diseases

Table 3. Examples of sequence homology of B27 binding CII peptides and bacterial peptide implicated in B27 associated diseases Amino acid sequence

Peptides derived from

1 IRLGAPQSL TRLGTAASL VRIGAPQSL VRLVVPSSL ERLAAASSL PRLTPPQPL

CII ŽG1070602; 2]10. S. typhimurium ŽP37168; 134]142. N. gonorrhoeae ŽP21302; 192]190. N. meningitidis ŽQ01325; 130]138. E. coli ŽP14633; 366]374. E. coli ŽP27304; 393]401. E.B. virus ŽP12978; 186]194.

2 GRTGPAGAA GRTGRAGRA

CII ŽG1070602; 333]341. K. pneumoniae ŽP33906; 350]358.

role in T cell selection, antigen presentation and peptide binding needs to be explored. Our studies suggest a role for CD4 q T cells and involvement of exogenous antigens. Is it possible that HC of B27

bind to exogenous antigens and triggers CD4 cells or effector CD8 cells with the help of CD4 lymphocytes. Further, studies with our HLA-B27 transgenic mouse model should reveal new insight into the role of the

Figure 1. Role of b 2 m-free heavy chains in T cell selection and presentation of exogenous antigens: a hypothesis. In the thymus: high affinity interaction of an endogenous peptide with mouse or human b 2 m associated HC of B27 will delete self-reactive T cells. However, the same autoreactive T cells may escape the negative selection when they interact with b 2 m-free heavy chains of HLA-B27. In the periphery: dissociation of b 2 m from heavy chains will lead to loose contact with endogenous peptide during antigen presentation. Loss of endogenously bound peptide to MHC molecules could allow binding of exogenous antigens. This process will lead to presentation of exogenous antigens by APCs to the self-reactive T cells.

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16. Urben RG, Chicz RM, Lane WS, Strominger JL, Rehm A, Kenter MJH, UytdeHaah FGCM, Ploegh H, Uchanska-Ziegler B, Ziegler A Ž1994. A subset of HLA-B27 molecules contains peptides much longer than nonamers. Proc Natl Acad Sci USA 91:1534]1538 17. Raghavan M, Lebron JA, Johnson JL, Bjorkman PJ Ž1996. Extended repertoire of permissible peptide ligands for HLABU 2702. Prot Sci 5:2080]2088 18. Lopez-Larrea C, Gonzalez-Roces S, Alvarez V Ž1996. HLA-B27 structure, function and disease association. Curr Opin Rheumatol 8:296]308 19. D’Amato M, Fiorillo MT, Carcassi C, Mathieu A, Zuccarelli A, Bitti PP, Tosi R, Sorrentino R Ž1995. Relevance of residue 116 of HLA-B27 in determining susceptibility to ankylosing spondylitis. Eur J Immunol 25:3199]3201 20. Fiorillo MT, Meadows L, D’Amato M, Shabanowitz J, Hunt DF, Appella E, Sorrentino R Ž1997. Susceptibility to ankylosing spondylitis correlates with the C-terminal residue of peptides presented by various HLA-B27 subtypes. Eur J Immunol 27:368]373 21. Yamaguchi A, Tsuchiya N, Mitsui H, Shiota M, Ogawa A, Tokunaga K, Yoshinoya S, Juji T, Ito K Ž1996. Association of HLA-B39 with HLA-B27-negative ankylosing spondylitis and pauciarticular juvenile rheumatoid arthritis in Japanese patients. Evidence for a role of the peptide-anchoring B pocket. Arthritis Rheum 39:1768]1769 22. Benjamin RJ, Madrigal JA, Parham P Ž1991. Peptide binding to empty HLA-B27 molecules of viable human cells. Nature 351:74]77. 23. Khare SD, Luthra HS, David CS Ž1996. Role of HLA-B27 in spondyloarthropathies. Curr Topics Microbiol Immunol 206:85]100 24. Scofield RH, Warren WL, Koelsch G, Harley JB Ž1993. A hypothesis for the HLA-B27 dysregulation in spondyloarthopathy: contribution from enteric organisms, B27 structure, peptides bound by B27, and convergent evolution. Proc Natl Acad Sci USA 90:9330]9334 25. Scofield RH, Kurien B, Gross T, Warren WL, Harley JB Ž1995. HLA-B27 binding of peptide from its own sequence and similar peptides from bacteria: implications for spondyloarthropathies. Lancet 345:1542]1544 26. Boisgerault F, Tieng V, Stolzenberg M-C, Dulphy N, Khalil I, Tamouza R, Charron D, Toubert A Ž1996. Differences in endogenous peptides presented by HLA-B U 2705 and BU 2703 allelic varients. J Clin Invest 98:2764]2770 27. Garcia F, Marina A, Albar JP, Lopez de Castro JA Ž1997. HLA-B27 presents a peptide from a polymorphic region of its own molecule with homology to proteins from arthritogenic bacteria. Tissue Antigens 49:23]28 28. Joyce S, Nathenson SG Ž1996. Alloreactivity, antigen recognition and T-cell selection: three diverse T-cell recognition problems with a common solution. Immunol Rev 154:59]103 29. Germain RN Ž1994. MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell 76:287]299 30. Rock KL, Gamble S, Rothstein L, Gramm C, Benacerraf B Ž1990. Dissociation of beta 2-microglobulin leads to the accumulation of a substantial pool of inactive class I MHC heavy chains on the cell surface. Cell 65:611]620. 31. Carreno BM, TH Hansen Ž1994. Exogenous peptide ligand influences the expression and half-life of free HLA class I heavy chains ubiquitously detected at the cell surface. Eur J Immunol 24:1285]1292 32. Sijts AJ, Pamer EG Ž1997. 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

B27 molecule in human disease pathogenesis. This transgenic animal model could also be used to investigate potential immunotherapy.

Acknowledgements Studies done in our laboratory were supported by an NIH grant AR39875.

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