Differential Association of HLA-B*2705 and B*2709 to Ankylosing Spondylitis Correlates with Limited Peptide Subsets but Not with Altered Cell Surface Stability

Abstract

In contrast to HLA-B*2705, B*2709 is weakly or not associated to ankylosing spondylitis. Both allotypes differ by a single D116H change. We compared the B*2705- and B*2709-bound peptide repertoires by mass spectrometry to quantify the effect of B*2709 polymorphism on peptide specificity. In addition, shared and differentially bound ligands were sequenced to define the structural features of the various peptide subsets. B*2705 shared 79% of its peptide repertoire with B*2709. Shared ligands accounted for 88% of the B*2709-bound repertoire. All B*2705 ligands not bound to B*2709 had C-terminal basic or Tyr residues. Most B*2709-bound peptides had C-terminal aliphatic and Phe residues, but two showed C-terminal Arg or Tyr. The B*2709-bound repertoire included 12% of peptides not found in B*2705. These had aliphatic C-terminal residues, which are also favored in B*2705. However, these peptides bound weakly B*2705 in vitro, indicating distinct contribution of secondary anchor residues in both subtypes. Differences in peptide binding did not affect the ratio of native to β2-microglobulin-free HLA-B27 heavy chain at the cell surface. Our results suggest that weaker association of B*2709 with ankylosing spondylitis is based on differential binding of a limited subset of natural ligands by this allotype. In contrast to HLA-B*2705, B*2709 is weakly or not associated to ankylosing spondylitis. Both allotypes differ by a single D116H change. We compared the B*2705- and B*2709-bound peptide repertoires by mass spectrometry to quantify the effect of B*2709 polymorphism on peptide specificity. In addition, shared and differentially bound ligands were sequenced to define the structural features of the various peptide subsets. B*2705 shared 79% of its peptide repertoire with B*2709. Shared ligands accounted for 88% of the B*2709-bound repertoire. All B*2705 ligands not bound to B*2709 had C-terminal basic or Tyr residues. Most B*2709-bound peptides had C-terminal aliphatic and Phe residues, but two showed C-terminal Arg or Tyr. The B*2709-bound repertoire included 12% of peptides not found in B*2705. These had aliphatic C-terminal residues, which are also favored in B*2705. However, these peptides bound weakly B*2705 in vitro, indicating distinct contribution of secondary anchor residues in both subtypes. Differences in peptide binding did not affect the ratio of native to β2-microglobulin-free HLA-B27 heavy chain at the cell surface. Our results suggest that weaker association of B*2709 with ankylosing spondylitis is based on differential binding of a limited subset of natural ligands by this allotype. ankylosing spondylitis spondyloarthropathies monoclonal antibody high pressure liquid chromatography matrix-assisted laser desorption/ionization time-of-flight mass spectrometry cytolytic T lymphocytes The association of HLA-B27 with ankylosing spondylitis (AS)1 and other spondyloarthropathies (Spa) ranks among the strongest of any MHC antigen with a human disease, although the mechanism of this association remains unknown. The differential linkage of HLA-B27 subtypes to AS revealed by population studies is expected to provide unique insight into the pathogenetic role of HLA-B27. Whereas B*2705, B*2702, B*2704, and B*2707 are all associated to AS (1Gonzalez-Roces S. Alvarez M.V. Gonzalez S. Dieye A. Makni H. Woodfield D.G. Housan L. Konenkov V. Abbadi M.C. Grunnet N. Coto E. Lopez-Larrea C. Tissue Antigens. 1997; 49: 116-123Crossref PubMed Scopus (209) Google Scholar), B*2706 and B*2709 are weakly or not associated to this disease (2Lopez-Larrea C. Sujirachato K. Mehra N.K. Chiewsilp P. Isarangkura D. Kanga U. Dominguez O. Coto E. Peña M. Setien F. Gonzalez-Roces S. Tissue Antigens. 1995; 45: 169-176Crossref PubMed Scopus (241) Google Scholar, 3Nasution A.R. Mardjuadi A. Kunmartini S. Suryadhana N.G. Setyohadi B. Sudarsono D. Lardy N.M. Feltkamp T.E. J. Rheumatol. 1997; 24: 1111-1114PubMed Google Scholar, 4Ren E.C. Koh W.H. Sim D. Boey M.L. Wee G.B. Chan S.H. Tissue Antigens. 1997; 49: 67-69Crossref PubMed Scopus (86) Google Scholar, 5D'Amato M. Fiorillo M.T. Carcassi C. Mathieu A. Zuccarelli A. Bitti P.P. Tosi R. Sorrentino R. Eur. J. Immunol. 1995; 25: 3199-3201Crossref PubMed Scopus (186) Google Scholar). In particular, B*2709 is not associated to AS in Sardinia, the only population where it has been found at significant frequency (5D'Amato M. Fiorillo M.T. Carcassi C. Mathieu A. Zuccarelli A. Bitti P.P. Tosi R. Sorrentino R. Eur. J. Immunol. 1995; 25: 3199-3201Crossref PubMed Scopus (186) Google Scholar). In continental Italy, where this allotype is found at much lower frequency (about 3% of HLA-B27 individuals), a patient with undifferentiated Spa without axial involvement was initially reported (6Olivieri I. Padula A. Cianco G. Moro L. Durante E. Guadiano C. Masciandaro S. Pozzi S. Ferrara G.B. Ann. Rheum. Dis. 2000; 59: 654-655Crossref PubMed Google Scholar). More recently, 2 of 47 individuals with Spa in southern continental Italy were found to be B*2709. Again, both patients showed undifferentiated Spa without axial involvement (7Olivieri I. Ciancio G. Padula A. Gaudiano C. Masciandaro S. Moro L. Durante E. Pozzi S. Ferrara G.B. Arthritis Rheum. 2002; 46: 553-554Crossref PubMed Scopus (25) Google Scholar). In an independent study (8Marchionni L. Modena V. Roggero R. Curtoni E.S. The polymorphism of HLA-B27 and the seronegative spondyloarthropathies in the Italian population.13th European Histocompatibility Conference. European Federation for Immunogenetics, Crete1999Google Scholar) a B*2709 individual with sacroiliitis and oligoarthritis was reported. The finding of a few individuals with Spa, but not AS, supports a lower association of B*2709 with this disease, while suggesting some involvement of this subtype with related forms of arthritis. B*2709 differs from B*2705 by a single amino acid substitution, D116H (9Del Porto P. D'Amato M. Fiorillo M.T. Tuosto L. Piccolella E. Sorrentino R. J. Immunol. 1994; 153: 3093-3100PubMed Google Scholar). This change, which is unique among known HLA-B27 subtypes, was reported to restrict peptide binding specificity to ligands with C-terminal nonpolar residues (10Fiorillo M.T. Meadows L. D'Amato M. Shabanowitz J. Hunt D.F. Apella E. Sorrentino R. Eur. J. Immunol. 1997; 27: 368-373Crossref PubMed Scopus (98) Google Scholar). This is in contrast with B*2705, which binds peptides with C-terminal basic, aliphatic, and aromatic residues (11Jardetzky T.S. Lane W.S. Robinson R.A. Madden D.R. Wiley D.C. Nature. 1991; 353: 326-329Crossref PubMed Scopus (735) Google Scholar). Previous studies did not address the effects of B*2709 polymorphism on other anchor positions of natural ligands or, more importantly, the overall effect of altered binding specificity on the constitutive peptide repertoire. In the present study we carried out a systematic comparison of the B*2705- and B*2709-bound peptide repertoires to determine the percentage of natural ligands shared by both allotypes. In addition, we sequenced a large number of peptides either common to both subtypes or selectively presented by one of them, to identify the structural features determining differential binding to both subtypes. Finally, we compared the expression of β2-microglobulin-free B*2705 and B*2709 heavy chains to test whether differential peptide binding might result in altered cell surface stability HLA B*2709 genomic DNA was obtained from a B*2705 genomic construct (12Calvo V. Rojo S. Lopez D. Galocha B. Lopez de Castro J.A. J. Immunol. 1990; 144: 4038-4045PubMed Google Scholar), substituting theXcmI-BssHII fragment (nucleotides 360–610) by the same fragment containing the 418G → C mutation (resulting in the D116H) obtained from a cDNA coding for B*2709 (a kind gift from R. Sorrentino). The resulting genomic construct was thoroughly sequenced to ensure that it contained only the desired mutation. HMy2.C1R (C1R) is a human lymphoid cell line with low expression of its endogenous class I antigens (13Storkus W.J. Howell D.N. Salter R.D. Dawson J.R. Cresswell P. J. Immunol. 1987; 138: 1657-1659PubMed Google Scholar, 14Zemmour J. Little A.M. Schendel D.J. Parham P. J. Immunol. 1992; 148: 1941-1948PubMed Google Scholar). B*2705-C1R transfectant cells were described elsewhere (12Calvo V. Rojo S. Lopez D. Galocha B. Lopez de Castro J.A. J. Immunol. 1990; 144: 4038-4045PubMed Google Scholar). The B*2709-C1R transfectant cell line was made as described (12Calvo V. Rojo S. Lopez D. Galocha B. Lopez de Castro J.A. J. Immunol. 1990; 144: 4038-4045PubMed Google Scholar) using the B*2709 gene. For some experiments a C1R cell line transfected with B*2709 cDNA was also used (15Garcia-Peydro M. Marti M. Lopez de Castro J.A. J. Immunol. 1999; 163: 2299-2305PubMed Google Scholar). C1R cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 7.5% fetal bovine serum or, for flow cytometry, in RPMI 1640 medium supplemented with 10% fetal bovine serum (all from Invitrogen). RMA-S is a TAP-deficient murine cell line (16Ljunggren H.G. Karre K. J. Exp. Med. 1985; 162: 1745-1759Crossref PubMed Scopus (647) Google Scholar, 17Townsend A. Ohlen C. Bastin J. Ljunggren H.G. Foster L. Karre K. Nature. 1989; 340: 443-448Crossref PubMed Scopus (884) Google Scholar). RMA-S transfectant cells expressing B*2705 and human β2-microglobulin have been previously described (18Villadangos J.A. Galocha B. Lopez de Castro J.A. J. Immunol. 1994; 152: 2317-2323PubMed Google Scholar). These cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. The mAbs used in this study were W6/32 (IgG2a, specific for a monomorphic HLA-A, B, C determinant) (19Barnstable C.J. Bodmer W.F. Brown G. Galfre G. Milstein C. Williams A.F. Ziegler A. Cell. 1978; 14: 9-20Abstract Full Text PDF PubMed Scopus (1600) Google Scholar), ME1 (IgG1, specific for HLA-B27, B7, Bw22) (20Ellis S.A. Taylor C. McMichael A. Hum. Immunol. 1982; 5: 49-59Crossref PubMed Scopus (201) Google Scholar), BBM.1 (IgG2b, specific for human β2-microglobulin) (21Brodsky F.M. Bodmer W.F. Parham P. Eur. J. Immunol. 1979; 9: 536-545Crossref PubMed Scopus (250) Google Scholar, 22Parham P. Androlewicz M.J. Holmes N.J. Rothenberg B.E. J. Biol. Chem. 1983; 258: 6179-6186Abstract Full Text PDF PubMed Google Scholar), and HC10 (IgG2a, specific for denatured and other forms of HLA class I heavy chain not associated to β2-microglobulin) (23Stam N.J. Spits H. Ploegh H.L. J. Immunol. 1986; 137: 2299-2306PubMed Google Scholar). About 6 × 104untransfected, B*2705- or B*2709-C1R transfectant cells were washed twice in 200 μl of phosphate-buffered saline and resuspended in 50 μl of undiluted mAb supernatant. After incubating for 30 min, cells were washed two times in 200 μl of phosphate-buffered saline and resuspended in 50 μl of fluorescein isothiocyanate-conjugated anti-mouse IgG rabbit antiserum (Calbiochem-Novabiochem GMBH, Schwalbach, Germany), incubated for 30 min, and washed two times in 200 μl of phosphate-buffered saline. All operations were done at 4 °C. Flow cytometry was carried out in an Epics Profile XL instrument (Coulter Electronics Inc., Hialeah, FL). Isolation of HLA-B27-bound peptides from about 1010 HLA-B27 transfectant C1R cells was carried out as previously described (24Paradela A. Garcia-Peydro M. Vazquez J. Rognan D. Lopez de Castro J.A. J. Immunol. 1998; 161: 5481-5490PubMed Google Scholar). Briefly, cells were lysed in 1% Nonidet P-40 in the presence of a mixture of protease inhibitors. After centrifugation, cell lysates were subjected to affinity chromatography using the W6/32 mAb. HLA-B27-bound peptides were eluted with 0.1% aqueous trifluoroacetic acid at room temperature, filtered trough Centricon 3 (Amicon, Beverly, MA), and concentrated for HPLC fractionation. This was conducted in a Waters Alliance system (Waters, Milford, MA) using a Vydac 218TP52 column (Vydac, Hesperia, CA) at a flow rate of 100 μl/min, exactly as described (25Paradela A. Alvarez I. Garcia-Peydro M. Sesma L. Ramos M. Vazquez J. Lopez de Castro J.A. J. Immunol. 2000; 164: 329-337Crossref PubMed Scopus (37) Google Scholar). Fractions of 50 μl were collected. The peptide composition of HPLC fractions was analyzed by MALDI-TOF MS using a calibrated Kompact Probe instrument (Kratos-Schimadzu) operating in the positive linear mode, as previously described (25Paradela A. Alvarez I. Garcia-Peydro M. Sesma L. Ramos M. Vazquez J. Lopez de Castro J.A. J. Immunol. 2000; 164: 329-337Crossref PubMed Scopus (37) Google Scholar). Dried fractions were resuspended in 5 μl of methanol/water (1:1) containing 0.1% formic acid, and 0.5-μl aliquots of the sample were deposited onto the stainless steel MALDI probe and allowed to at room μl of acid in aqueous and 0.1% trifluoroacetic was and allowed to at room was carried out by in an instrument exactly as elsewhere J. Vazquez J. Lopez de Castro J.A. Tissue Antigens. 1998; PubMed Scopus Google Scholar, A. J. Lopez de Castro J.A. Vazquez J. J. 1999; PubMed Scopus Google Scholar). peptide was sequenced by using the with The HPLC was and in as was with μl of in trifluoroacetic Full MS was carried out by and the was subjected to of the or residues was done on the of with known in the In one of was not by this and the was on the of of in HPLC of the but not of the peptide with with the natural These were using and by The mass of peptides was by MALDI-TOF and composition and was done by amino acid in using a The used to peptide binding was as described B. J.R. J.A. Lopez de Castro J.A. Tissue Antigens. PubMed Scopus (37) Google Scholar) with Briefly, B*2705 RMA-S were incubated at for in RPMI 1640 medium supplemented with 10% fetal bovine serum. These were washed times in medium incubated for at with various peptide in the same to and for flow cytometry 4 HLA-B27 expression was using 50 μl of containing the mAb of the used as positive was as which is the of the peptide at of the obtained at the of other peptides was as the of peptide to the at the of the This was as The B*2705- and B*2709-bound peptide were from the C1R transfectant cells of HLA-B27 and acid The peptide were by HPLC and the peptide composition of individual fractions was analyzed by MALDI-TOF The of HPLC from one subtype was compared with the and fractions from the other In this comparison only or more of the in the HPLC of at one subtype were into with the same ratio and times were as ligands in both subtypes. of the of the peptide mixture analyzed this is not the it is to that the of peptides with the same mass and from these peptide are This was by peptides from both subtypes in from only one subtype in two independent were as peptides differentially bound to that In a number of a in one subtype had a much in the other In all these the peptides were as shared the showed of the in the MALDI-TOF MS and the of its in the other subtype was at lower in two independent the peptide was as a This of be with the of a peptide in have significant and is not an of peptide However, with the and we to in for this of and were compared from the B*2705- and B*2709-bound peptide were in both subtypes, only in B*2705, and only in B*2709. the peptide of peptides not found in B*2709. the B*2709-bound peptide about 12% of peptides from B*2705. The mass of B*2705 and B*2709 ligands was although peptides differentially bound to B*2705 had a mass the shared ligands and from B*2705 or showed to the for were as in B*2705, and in of B*2705- and B*2709-bound peptide B*2705 and in a These results that the single amino acid B*2709 from B*2705 binding of 79% of the natural binding of the and the of bound peptide of at 10% of shared B*2709 polymorphism to this subtype distinct binding that 12% of the peptide repertoire from this subtype is from B*2705, and at of shared ligands are more in B*2709. of peptides were by from the B*2705- B*2709-bound peptide included shared of which were in B*2705 (2Lopez-Larrea C. Sujirachato K. Mehra N.K. Chiewsilp P. Isarangkura D. Kanga U. Dominguez O. Coto E. Peña M. Setien F. Gonzalez-Roces S. Tissue Antigens. 1995; 45: 169-176Crossref PubMed Scopus (241) Google Scholar) or B*2709 (5D'Amato M. Fiorillo M.T. Carcassi C. Mathieu A. Zuccarelli A. Bitti P.P. Tosi R. Sorrentino R. Eur. J. Immunol. 1995; 25: 3199-3201Crossref PubMed Scopus (186) Google Scholar), ligands found only in B*2705, and ligands found only in B*2709 All the peptides found only in B*2705 had C-terminal basic or Tyr residues, shared had C-terminal nonpolar residues, aliphatic or These results studies (10Fiorillo M.T. Meadows L. D'Amato M. Shabanowitz J. Hunt D.F. Apella E. Sorrentino R. Eur. J. Immunol. 1997; 27: 368-373Crossref PubMed Scopus (98) Google Scholar) that B*2709 polymorphism binding to peptides with C-terminal residues. In addition, two from this was not two peptides with C-terminal Tyr and were found as natural ligands of in much lower in B*2705. The of the peptide with a C-terminal Tyr in B*2709 was by a number of peptides with C-terminal aliphatic residues were found only or in indicating although these residues are also favored in B*2705, some peptides not or to this subtype in However, differential of residues was at positions other the C-terminal one peptides found only or in B*2705, and differentially bound to B*2709. that B*2709 ligands not found in B*2705 were to this peptides were for binding to B*2705 in an using RMA-S transfectant cells All B*2709 ligands bound B*2705 with and more weakly a B*2705 used as positive 3 These results anchor residues for B*2705, some B*2709 ligands are not found in B*2705. We the that in the and affinity of the constitutive peptide repertoire by B*2709 polymorphism might the expression of HLA-B27 heavy chain on the cell as with the mutation on B*2705 I. M. Vazquez J. E. S. Lopez de Castro J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (37) Google Scholar). HLA-B27 expression was analyzed on B*2705- and B*2709-C1R transfectant cells by flow cytometry with mAbs and all with the native and with which with β2-microglobulin-free HLA class I heavy all mAbs the surface expression of either native or β2-microglobulin-free B*2705 and B*2709 was The ratio of to was about and for B*2705 and suggesting that about of the of subtype were at the cell surface as β2-microglobulin-free heavy of B*2705 and B*2709 on the surface of C1R transfectant in a The HLA-B27 B*2705, binds with few I. M. Vazquez J. E. S. Lopez de Castro J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (37) Google Scholar) peptides with an and C-terminal basic, aliphatic, or aromatic residues (11Jardetzky T.S. Lane W.S. Robinson R.A. Madden D.R. Wiley D.C. Nature. 1991; 353: 326-329Crossref PubMed Scopus (735) Google Scholar). Whereas is a shared with other HLA-B27 subtypes, these differ among other in the of C-terminal residues. B*2702, which B*2705 is associated to AS only peptides with C-terminal aliphatic or aromatic residues O. K. S. V. G. H.G. 1994; PubMed Scopus Google Scholar), it was that HLA-B27 ligands with C-terminal basic residues not be for to The that B*2706 and B*2709 did not peptides with C-terminal a that is by subtypes B*2705, B*2702, and B*2704, that peptides might this (10Fiorillo M.T. Meadows L. D'Amato M. Shabanowitz J. Hunt D.F. Apella E. Sorrentino R. Eur. J. Immunol. 1997; 27: 368-373Crossref PubMed Scopus (98) Google Scholar, F. A. Lopez de Castro J.A. Tissue Antigens. 1997; 49: PubMed Scopus Google Scholar). This was by the finding that the subtype B*2707 to a C-terminal Tyr V. N. F. R. D. A. 1997; PubMed Scopus Google Scholar). to peptides with C-terminal Tyr was for B*2706 L. V. J.R. A. Vazquez J. Lopez de Castro J.A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) in the present for but were revealed by results although B*2709 specificity is to peptides with C-terminal aliphatic or Phe residues two peptides with C-terminal Arg and were found in the B*2709-bound peptide both peptides were much more in B*2705 in B*2709 be significant MS were which are known to of peptides presented at the cell surface. of peptides with C-terminal basic or Tyr residues by B*2709 is not some B*2709 ligands with C-terminal aliphatic residues were not found in B*2705 and bound this subtype in with although B*2705 peptides with these was differential features from the of binding of some peptides with C-terminal nonpolar residues to B*2709 be of a of at secondary anchor This be with binding only by of the C-terminal nonpolar in the more of B*2709. B*2709 polymorphism might at secondary anchor although this effect be the effect on C-terminal and its might high of individual peptide the ratio cell and β2-microglobulin-free heavy chains was the same for both subtypes. We have previously I. M. Vazquez J. E. S. Lopez de Castro J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (37) Google Scholar) that of this ratio the in an HLA-B27 with lower overall affinity for the peptide repertoire. results suggest that B*2705 and B*2709 not differ in the stability of the at the cell surface. in this study was to the in C-terminal by B*2709 polymorphism altered the peptide to B*2705. All sequenced not found in B*2709 had C-terminal or indicating that the subset of peptides with these C-terminal residues is from B*2709. In B*2705 shared about 79% of its peptide repertoire with B*2709. This is with the of of with reported previously (15Garcia-Peydro M. Marti M. Lopez de Castro J.A. J. Immunol. 1999; 163: 2299-2305PubMed Google Scholar). This that shared ligands and features bound to both subtypes. The of results for the pathogenetic role of HLA-B27 in Spa is on the to which B*2709 determine for this of this subtype has been found with significant frequency only in Sardinia, the strongest involvement of this allotype in AS from the of B*2709 AS patients in this population (5D'Amato M. Fiorillo M.T. Carcassi C. Mathieu A. Zuccarelli A. Bitti P.P. Tosi R. Sorrentino R. Eur. J. Immunol. 1995; 25: 3199-3201Crossref PubMed Scopus (186) Google Scholar). of the reported B*2709 individuals with Spa in continental Italy showed AS or axial for sacroiliitis in one (7Olivieri I. Ciancio G. Padula A. Gaudiano C. Masciandaro S. Moro L. Durante E. Pozzi S. Ferrara G.B. Arthritis Rheum. 2002; 46: 553-554Crossref PubMed Scopus (25) Google Scholar), supports that B*2709 is weakly or not associated with AS but also that this subtype to related forms of arthritis. P. McMichael A. 1999; PubMed Scopus Google Scholar, P. J.R. Immunol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, R.A. Med. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), results are with involvement of some to a limited of the repertoire in the of AS, as by the peptide R. Parham P. Immunol. 1990; Full Text PDF PubMed Scopus Google Scholar). peptides with C-terminal basic residues are not bound to B*2702, results in with studies (10Fiorillo M.T. Meadows L. D'Amato M. Shabanowitz J. Hunt D.F. Apella E. Sorrentino R. Eur. J. Immunol. 1997; 27: 368-373Crossref PubMed Scopus (98) Google Scholar, F. A. Lopez de Castro J.A. Tissue Antigens. 1997; 49: PubMed Scopus Google Scholar, L. V. J.R. A. Vazquez J. Lopez de Castro J.A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), that might have C-terminal Tyr. The of B*2709 to low of some peptides with this might the of this subtype with undifferentiated The that the B*2707 subtype not peptides with C-terminal based on by V. N. F. R. D. A. 1997; PubMed Scopus Google Scholar) might using the much more MS to determine whether peptides might B*2707 in as found for B*2709. is an to the the B*2705- and B*2709-bound peptide repertoires and the differential association of these subtypes to is that a peptide might to a subtype not associated to AS to a of peptide be favored for the subtype to present it more For B*2705 and this was by a study that a peptide from a that in AS patients on individuals bound B*2709 B*2705 M.T. M. R. M.L. Sorrentino R. J. 2000; PubMed Scopus Google Scholar). to this peptides be among with C-terminal nonpolar residues that are selectively or presented by as found in this both but results to on the limited of HLA-B27-bound peptides that are not among subtypes differentially associated to We Sorrentino for the gift of B*2709 cDNA and for in We also for