The same natural ligand is involved in allorecognition of multiple HLA-B27 subtypes by a single T cell clone: role of peptide and the MHC molecule in alloreactivity

The human alloreactive CTL clone 27S69, raised against B*2705, cross-reacts with B*2702 and B*2703, but not with B*2701, B*2704, B*2706, or B*2710. Its natural epitope was identified by electrospray/ion trap mass spectrometry, as the proteasome-derived RRFFPYYV octamer. This is the first HLA-B27 ligand shown to be immunogenic in alloreactivity. The RRFFPYYVY nonamer, also found in the B*2705-bound peptide pool, was recognized much less efficiently, demonstrating that an alloreactive CTL distinguishes between very similar natural ligands. Molecular modeling suggested that this was due to the different conformation of each peptide in complex with B*2705. B*2702- and B*2703-RMA-S cells were lysed by CTL 27S69 when sensitized with the octamer, demonstrating that cross-reaction with these subtypes is through recognition of the same peptide as in B*2705. B*2704-, B*2706-, and B*2710-RMA-S cells were not sensitized for lysis, in spite of efficient binding of the octamer, indicating that polymorphism in these subtypes directly impairs allorecognition. B*2701-RMA-S and -C1R cells were sensitized for lysis by the octamer, suggesting lack of the endogenous peptide epitope on this subtype. Absence of the octamer in the B*2701-bound peptide pool further suggested that B*2701 polymorphism impairs the generation of this peptide.     

Binding of peptides naturally presented by HLA-B27 to the differentially disease-associated B*2704 and B*2706 subtypes, and to mutants mimicking their polymorphism

B*2704 and B*2706 are closely related HLA-B27 subtypes of which the former but not the latter is associated to ankylosing spondylitis. Their peptide specificity relative to other disease-associated subtypes was analyzed by testing binding of self-peptides naturally presented by B*2705 or B*2702, and synthetic analogs, to B*2704, B*2706, and site-specific mutants mimicking their changes. Peptides with basic, aliphatic or aromatic C-terminal residues bound to B*2705 with similar affinity. In B*2704 C-terminal aliphatic/ aromatic residues were preferred. B*2706 discriminated drastically between polar and nonpolar C-terminal residues, showing strong preference for Leu and Phe, and less than B*2704 for basic and Tyr residues. Loss of single acidic charges (D > S77, D > Y116) increased preference for C-terminal Leu and Phe, but allowed efficient binding of peptides with basic residues or Tyr. Their gain (V > E152, H > D114) maintained wide C-terminal specificity, but severely impaired binding, presumably by disrupting interactions with internal peptide residues. This was compensated by Y116 in the double D114Y116 mutant. The specificity of B*2704 and B*2706 was explained only partially by the separate effects of single mutations, indicating that novel properties arise from concomitant changes at various positions. For instance, specificity of B*2706 for nonpolar C-terminal residues required simultaneous removal of Asp77 and Asp116. B*2706 differed from B*2705, B*2702, and B*2704 in its lower suitability for C-terminal Tyr, suggesting that this feature might be relevant for HLA-B27 association to spondyloarthropathy.     

Routine molecular genotyping of HLA-B27 in spondyloarthropathies overcomes the obstacles of serological typing and reveals an increased B *2702 frequency in ankylosing spondylitis

OBJECTIVE: To evaluate the reproducibility and reliability of polymerase chain reaction (PCR) in HLA-B27 typing compared to the conventionally used microlymphocytotoxicity test (MLCT). To determine the HLA-B27 subtype frequencies (B*2701-B*2709) in patients with HLA-B27 associated disease and healthy persons using sequence specific oligonucleotides (SSO). METHODS: 398 consecutive patients were HLA-B27 typed by MLCT and PCR. Subtyping by SSO was performed in 142 patients with HLA-B27 associated disease [ankylosing spondylitis (AS) n = 38, reactive arthritis 44, undifferentiated spondyloarthropathy (uSpA) 45, psoriatic arthritis 15] and 125 healthy HLA-B27 controls. RESULTS: MLCT identified 61 HLA-B27 positive patients (15.3%); PCR identified 78 positive patients (19.6%). MLCT gave false negative results for 8 patients (2.0%) and false positives for a further 7 (1.8%). Only subtypes B*2702 and B*2705 were present in patients and controls. Overall frequencies of B*2702 in patients and controls were 14.1 and 9.6%, respectively. The B*2702 frequency was significantly (pcorr. < 0.04) higher in AS (23.7%) and lower in uSpA (6.7%) patients. CONCLUSION: HLA-B27 typing by PCR is reliable and reproducible and therefore recommended for routine typing. It overcomes the obstacles of serological typing, i.e., equivocal results and cross-reactivity. In addition, subtype frequencies (B*2702 and B*2705) are equally distributed among patients and controls, although subtype B*2702 seems to be more frequent in AS and less so in uSpA.     

Evaluation of a corporate-sponsored health care program for retired employees

Five HLA-B27 subtypes, B*2701, B*2703, B*2704, B*2705, and B*2706, were tested for direct binding with twenty-six synthetic nonapeptides carrying the primary anchor residue motifs (combination of amino residues at positions 2 and 9) relevant to B*2705. The peptide sequences were derived from human HSP89 alpha, P53 and MBP. The alpha chains were immunospecifically isolated from LH (B*2701), CH (B*2703), WE1 (B*2704), BTB (B*2705), and LIE (B*2706) cells and their peptide binding was measured by the HLA class I alpha chain refolding assay. The data obtained indicated that the B27 subtypes tested can bind a common set of peptides carrying several different anchor residue motifs. The motifs, R-K and R-R, reported for B*2705 and a new motif H-R were accepted by B*2703, B*2704, and B*2706, but not by B*2701. However, other motifs, including known B*2702 and/or B*2705 motifs, R-H, R-L, R-A, and R-F, and a new motif found here, R-G, were apparently accepted by all B27 subtypes tested. The observed cross-peptide binding in the B27 subgroup is compatible with the so-called arthritogenic peptide hypothesis in the pathogenesis of ankylosing spondylitis.     

Overlapping peptide-binding specificities of HLA-B27 and B39: evidence for a role of peptide supermotif in the pathogenesis of spondylarthropathies

OBJECTIVE: Previous studies indicated the increase of HLA-B39 among HLA-B27 negative patients with spondylarthropathies (SpA). This study was performed to examine whether the natural ligands of HLA-B27 are capable of binding to HLA-B39. METHODS: Peptides were synthesized according to the sequences of known natural ligands of HLA-B27 or B39 and were tested for their binding to HLA-B*3901 and B*2705 by quantitative peptide binding assay, using a TAP-deficient RMA-S cell line transfected with human beta2-microglobulin and HLA class I heavy chain genes. RESULTS: Four of the 10 HLA-B27 binding peptides significantly bound to HLA-B*3901. All 4 peptides had hydrophobic/aromatic amino acids (Leu or Phe) at the C-terminus. In contrast, peptides with basic residues (Lys, Arg) or Tyr at the C-terminus did not bind to B*3901. In parallel experiments, 1 of the 2 natural ligands of HLA-B*3901 was found to bind to B*2705. CONCLUSION: A subset of natural HLA-B27 ligands was capable of binding to B*3901. In addition to Arg at position 2 (Arg2), hydrophobic/aromatic C-terminal residues, such as Leu or Phe, seemed to be crucial for the cross-specificity. These results suggested that HLA-B27 and B39 recognize overlapping peptide repertoires, supporting the hypothesis that the peptides presented by both of these class I antigens play a role in the pathogenesis of SpA.     

Transformation from SAA2-fibrils to AA-fibrils in amyloid fibrillogenesis: in vivo observations in murine spleen using anti-SAA and anti-AA antibodies

PCR in combination with SSO probes was used to analyze the polymorphism in exons 2 and 3 of HLA-B27 subtypes and HLA-C-related alleles in two genetically distant Caucasian groups: Spanish and Jewish populations. AS patients and healthy B27 donors from both populations were analyzed in order to ascertain B27-Cw haplotypes. Three different ancestral haplotypes were found to be represented in both populations: B*2705/Cw*0102, B*2705/Cw*02022, and B*2702/Cw*02022. The B*2705 (92.5%) was the most frequent allele found in the Spanish population, carried by B*2705/Cw*0102 (60.9%) and B2705/Cw*02022 (30.4%) haplotypes. In contrast, B*2702 (59.4%) was the most prevalent allele found in the Jewish population and was carried by the B*2702/Cw*02022 (63.3%) haplotype. No different allelic and haplotypic distributions were among healthy and AS patients in either Spanish or Jewish populations. The differences found in the distribution of B27 haplotypes among Spanish and Jewish Caucasian populations are consistent with the genetic distance of these ethnic groups. When the Jewish population was subdivided into Ashkenazi (A) and non-Ashkenazi (NA) groups, no significant differences were observed in the distribution of B*2702/Cw*02022 haplotype. Minor differences were observed in the underrepresented B*2705 haplotypes. The present results reflect the ancestral affinities of A and NA Jewish populations. A possible HLA-B27 evolutive pathway in Caucasians is proposed according to the data available for the B27/Cw ancestral haplotypes in Spanish and Jewish groups.     

Spondyloarthropathies and HLA-B27 subtypes in the populations of the Russian North

The aim of the study was to investigate the role of HLA-B27 subtypes in development of ankylosing spondylitis and other seronegative spondylarthropathies. Using oligotyping techniques we studied native DNA of 219 HLA-B27 positive natives: 88 Chukotka residents and 131 Mordovians (Russian Ugro-Finnish population). Only subtypes HLA-B*2705 and B*2702 were revealed. A dominant subtype of HLA-B27 among the natives was HLA-B*2705: 99% among residents of Chukotka and 86% among Mordovians. It was established that among spondylarthropathic patients the frequency of B*2705 does not differ from its incidence in the studied populations. The data support the suggestion that several B27 subtypes and common genetic determinant of B27 gene may be involved in pathogenesis of spondylarthropathy.     

Stenotic origin of an aberrant left subclavian artery from a right-sided aortic arch. A case report

OBJECTIVE: To refine the algorithms governing peptide presentation by HLA-B*2705 by analyzing: (i) the specificity of the human transporter associated with antigen processing (TAP) for HLA-B27 binding peptides; and (ii) the peptide binding affinity to HLA-B*2705. METHODS: TAP-translocation was measured with a labeled reporter peptide containing an N-linked glycosylation acceptor site in Streptolysin O-permeabilized cells for a panel of HLA-B27 binding peptides. Peptide binding affinity was determined by peptide-induced stabilization of empty HLA-B*2705 expressed by the TAP-deficient cell line T2-B27. RESULTS: Human TAP preferentially translocated analogues with residues leucine, isoleucine, methionine and arginine as the carboxy-terminal amino acids, whereas analogues with aspartic acid and serine were translocated poorly. The binding affinity to HLA-B*2705 of the poorly translocated aspartic acid and serine analogues was about 100-fold less compared to the parent HLA-B27 binding peptide. CONCLUSIONS: Human TAP shows considerable specificity for the C-terminus of potential HLA-B27 ligands. Nonamer peptides with aspartic acid and serine at the C-terminus are poorly translocated by the TAP and have low binding affinity for HLA-B*2705, and are therefore unlikely to become presented by HLA-B*2705.     

Speaking behavior and speech sound characteristics in acute schizophrenia

Analysis of peptides derived from HLA class I molecules indicates that thousands of unique peptides are bound by a single molecular type, and sequence examination of the pooled constituents yields a motif which collectively defines the peptides bound by a given class I molecule. Motifs resulting from pooled sequencing are then used to infer whether particular viral and tumor protein fragments might serve as class I-presented peptide therapeutics. Still undetermined from a pooled motif is the breadth or range of peptides in the population which are brought together to form the pooled motif, and it is therefore not yet known how representative of the population a pooled motif is. By employing hollow fiber bioreactors for large-scale production of HLA class I molecules, sufficient peptides are produced to investigate individual subsets of peptides comprising a motif. Edman sequencing and mass spectrometric analysis of peptides eluted from HLA-B*1501 reveal that many peptide sequences fail to align with either the N- or C-terminal anchors predicted for the B*1501 peptide motif through whole pool sequencing. These analyses further reveal auxiliary anchors not previously detected and peptides significantly larger and smaller than the predicted nonamer, ranging from 6 to 12 amino acids in length. These results demonstrate that constituents of the B*1501 peptide pool vary markedly in comparison with one another and therefore in comparison with previously established B*1501 motifs, and such complexity indicates that many of the peptide ligands presented to CTL cannot be predicted using class I consensus motifs as search criteria.     

A single position in the third transmembrane domains of the human B1 and B2 bradykinin receptors is adjacent to and discriminates between the C-terminal residues of subtype-selective ligands

In order to identify agonist- and antagonist-binding epitopes in the human B1 and B2 bradykinin (BK) receptors, we exploited the ability of these receptors to discriminate between peptide ligands that differ only by the absence (B1) and presence (B2) of a C-terminal Arg. This was done by constructing chimeric proteins in which specific domains were exchanged between these receptors as recently described by us (Leeb, T., Mathis, S. A., and Leeb-Lundberg, L. M. F. (1997) J. Biol. Chem. 272, 311-317). The constructs were then expressed in HEK293 and A10 cells and assayed by radioligand binding and by agonist-stimulated inositol phospholipid hydrolysis and intracellular Ca2+ mobilization. Substitution of the third transmembrane domain (TM-III) of the B1 receptor in the B2 receptor (B2(B1III)) dramatically reduced the affinities of B2-selective peptide ligands including both the agonist BK and the antagonist NPC17731. High affinity binding of both ligands to B2(B1III) was fully regained when one residue, Lys111, in TM-III of this chimera was replaced with the corresponding wild-type (WT) B2 receptor residue, Ser (B2(B1IIIS111)). Replacement of Ser111 with Lys in the WT B2 receptor decreased the affinities of BK and NPC17731 and increased the affinity of the B1-selective des-Arg10 analog of NPC17731, NPC18565. The results show that the C-terminal residue of peptide agonists and antagonists when bound to the B2 receptor is adjacent to Ser111 in the receptor. A Lys at this position, as is the case in the WT B1 receptor, provides a positive charge that repels the C-terminal Arg in B2-selective peptides and attracts the negative charge of the C terminus of B1-selective peptides, which lack the C-terminal Arg. Therefore, the residues at this one single position are crucial in determining the peptide selectivity of B1 and B2 BK receptors.     

Molecular basis for substrate specificity of protein-tyrosine phosphatase 1B

Protein-tyrosine phosphatases can exhibit stringent substrate specificity in vivo, although the molecular basis for this is not well understood. The three-dimensional structure of the catalytically inactive protein-tyrosine phosphate 1B (PTP1B)/C215S complexed with an optimal substrate, DADEpYL-NH2, reveals specific interactions between amino acid residues in the substrate and PTP1B. The goal of this work is to rigorously evaluate the functional significance of Tyr46, Arg47, Asp48, Phe182, and Gln262 in substrate binding and catalysis, using site-directed mutagenesis. Combined with structural information, kinetic analysis of the wild type and mutant PTP1B using p-nitrophenyl phosphate and phosphotyrosine-containing peptides has yielded further insight into PTP1B residues, which recognize general features, as well as specific properties, in peptide substrates. In addition, the kinetic results suggest roles of these residues in E-P hydrolysis, which are not obvious from the structure of PTP1B/peptide complex. Thus, Tyr46 and Asp48 recognize common features of peptide substrates and are important for peptide substrate binding and/or E-P formation. Arg47 acts as a determinant of substrate specificity and is responsible for the modest preference of PTP1B for acidic residues NH2-terminal to phosphotyrosine. Phe182 and the invariant Gln262 are not only important for substrate binding and/or E-P formation but also important for the E-P hydrolysis step.     

Importance of a conserved TCR J alpha-encoded tyrosine for T cell recognition of an HLA B27/peptide complex

Human HLA B27-restricted cytotoxic T lymphocytes (CTL) specific for the influenza A epitope NP383-391 use similar TCR alpha and beta chains, with two closely related J alpha segments used by six of nine CTL clones from three unrelated donors (Bowness et al., Eur J. Immunol. 1993. 23: 1417-1421). The role of TCR complementarity-determining region (CDR)3alpha residues 93 and 100-102 was examined by site-directed mutagenesis, following expression of the TCR alpha and beta extracellular domains from one clone as a TCR zeta fusion heterodimer in rat basophil leukemia (RBL) cells. For the first time we have measured direct binding of tetrameric HLA B*2705/NP383-391 complexes to transfected TCR. Independently peptide-pulsed antigen-presenting cells (APC) were used to induce TCR-mediated degranulation of RBL transfectants. Our results show a key role for the conserved TCRalpha CDR3 J alpha-encoded residue Y102 in recognition of HLA B27/NP383-391. Thus the Y102D mutation abolished both tetramer binding and degranulation in the presence of peptide-pulsed APC. Even the Y102F mutation, differing only by a single hydroxyl group from the native TCR, abolished detectable degranulation. Further mutations F93A and S100R also abolished recognition. Interestingly, the N101A mutation recognized HLA B27/NP in functional assays despite having significantly reduced tetramer binding, a finding consistent with "kinetic editing" models of T cell activation. Modeling of the GRb TCR CDR3alpha loop suggests that residue Y102 contacts the HLA B*2705 alpha1 helix. It is thus possible that selection of germ-line TCRAJ-encoded residues at position 102 may be MHC driven.     

HLA-B27 molecular subtypes and spondylarthropathies

The close association between HLA-B27 and spondyloarthropathies remains unexplained. Twelve HLA-B27 subtypes designated B*2701 to B*2712 have been described in various populations. Variations in the ability of these alleles to carry susceptibility to spondyloarthropathies may exist, and may be ascribable to differences in endogenous peptide presentation. This hypothesis was evaluated by a study of peptide-binding motifs of endogenous peptides extracted from various HLA-B27 alleles. A peptide motif with a tyrosine residue at the C-terminus was not characteristic of HLA-B27 subtypes carrying susceptibility, consistent with the known lack of association of B*2707 with spondyloarthropathies. However, at the level of the individual, differences may exist between endogenous peptides presented by subtypes that do and do not confer susceptibility.     

Histophysiological observations on the external auditory meatus, middle, and inner ear of the Weddell seal (Leptonychotes weddelli)

The assembly assay for peptide binding to class I major histocompatibility complex (MHC) is based on the ability to stabilise MHC class I molecules from mutant cell lines by the addition of suitable peptides. Such cell lines lack a functional transporter associated with antigen presentation (TAP) and as a result accumulate empty, unstable class I molecules in the ER. These dissociate rapidly in cell lysates unless they are stabilised by the addition of an appropriate binding peptide during lysis. The extent of stabilisation of class I molecules is directly related to the binding affinity of the added peptide. However, some MHC class I molecules, including HLA-B * 2705 and H-2Kk are unusually stable in their peptide-receptive state making them inappropriate for analysis using this assay or assays which depend on the ability of peptides to stabilise MHC class I molecules at the cell surface. Here we present an improved method that permits reliable measurements of peptide binding to such class I MHC molecules that are unusually stable in the absence of peptide. Cells are lysed in the presence of peptide and incubated at 4 degrees C. After 2 h, during which peptide binding to empty MHC molecules occurs, the lysate is heated to a temperature which preferentially destabilises those MHC molecules that remain empty. We have used this technique to assay peptide binding to HLA-B * 2705, as well as to the murine allele H-2Kk which also displays a stable phenotype when transfected into TAP-deficient T2 cells and show that this method represents a marked improvement over previous methods in terms of lower background signal and higher recovery of peptide bound molecules.     

Mutations of androgen receptor gene in Brazilian patients with male pseudohermaphroditism

We describe the identification of point mutations in the androgen receptor gene in five Brazilian patients with female assignment and behavior. The eight exons of the gene were amplified by the polymerase chain reaction (PCR) and analyzed for single-strand conformation polymorphism (SSCP) to detect the mutations. Direct sequencing of the mutant PCR products demonstrated single transitions in three of these cases: G-->A in case 1, within exon C, changing codon 615 from Arg to His; G-->A in case 2, within exon E, changing codon 752 from Arg to Gln, and C-->T in case 3, within exon B, but without amino acid change.     

A PCR-based method for uniform 13C/15N labeling of long DNA oligomers

p53 is very often mutated in human cancers. The majority of alterations are missense mutations located within the DNA-binding domain of the protein. Many reports have characterized such mutant proteins. Little is known, however, about the properties of proteins that have a missense mutation outside this domain. We investigated here the properties of 8 mutant proteins identified in human tumors as having a missense mutation in the tetramerization domain. The Arg342Gln, Glu349Asp and Gln354Arg proteins behaved like wild-type both in vitro and in cells. Two mutants, Arg342Pro and Leu344Pro, were inactive in all assays. Finally, the 3 mutant proteins Leu330His, Arg337Cys and Arg337Leu, which are inactive in vitro, showed no activity at low expression levels in cells but became active at higher expression levels. Our results reveal new phenotypes for p53 mutants and suggest that sequencing of the p53 gene from patients with tumors should be extended to exons 9 and 10 in clinical investigations.     

HLA-B27-restricted antigen presentation in the absence of tapasin reveals polymorphism in mechanisms of HLA class I peptide loading

Tapasin is a resident ER protein believed to be critical for antigen presentation by HLA class I molecules. We demonstrate that allelic variation in MHC class I molecules influences their dependence on tapasin for peptide loading and antigen presentation. HLA-B*2705 molecules achieve high levels of surface expression and present specific viral peptides in the absence of tapasin. In contrast, HLA-B*4402 molecules are highly dependent upon human tapasin for these functions, while HLA-B8 molecules are intermediate in this regard. Significantly, HLA-B*2705 like HLA-B*4402, requires tapasin to associate efficiently with TAP (transporters associated with antigen processing). The unusual ability of HLA-B*2705 to form peptide complexes without associating with TAP or tapasin confers flexibility in the repertoire of peptides presented by this molecule. We speculate that these properties might contribute to the role of HLA-B27 in conferring susceptibility to inflammatory spondyloarthropathies.     

Structures that delineate orphanin FQ and dynorphin A pharmacological selectivities

A genetic polymorphism in the metabolism of the anticonvulsant drug S-mephenytoin has been attributed to defective CYP2C19 alleles. This genetic polymorphism displays large interracial differences with the poor metabolizer (PM) phenotype representing 2-5% of Caucasian and 13-23% of Oriental populations. In the present study, we identified two new mutations in CYP2C19 in a single Swiss Caucasian PM outlier (JOB 1) whose apparent genotype (CYP2C19*1/CYP2C19*2) did not agree with his PM phenotype. These mutations consisted of a single base pair mutation (G395A) in exon 3 resulting in an Arg132-->Gln coding change and a (G276C) mutation in exon 2 resulting in a coding change Glu92-->Asp. However, the G276C mutation and the G395A mutation resided on separate alleles. Genotyping tests of a family study of JOB1 showed that the exon 2 change occurred on the CYP2C19*2 allele, which also contained the known splice mutation in exon 5 (this variant is termed CYP2C19*2B to distinguish it from the original splice variant now termed CYP2C19*2A). The exon 3 mutation resided on a separate allele (termed CYP2C19*6). In all other respects this allele was identical to one of two wild-type alleles, CYP2C19*1B. The incidence of CYP2C19*6 in a European Caucasian population phenotyped for mephenytoin metabolism was 0/344 (99% confidence limits of 0 to 0.9%). Seven of 46 Caucasian CYP2C19*2 alleles were CYP2C19*2B(15%) and 85% were CYP2C19*2A. The Arg132Gln mutation was produced by site-directed mutatgenesis and the recombinant protein expressed in a bacterial cDNA expression system. Recombinant CYP2C19 6 had negligible catalytic activity toward S-mephenytoin compared with CYP2C19 1B, which is consistent with the conclusion that CYP2C19*6 represents a PM allele. Thus, the new CYP2C19*6 allele contributes to the PM phenotype in Caucasians.     

Alteration of HLA-B27 peptide presentation after infection of transfected murine L cells by Shigella flexneri

Shigella flexneri is a triggering agent for reactive arthritis in HLA-B27-susceptible individuals. Considering the intracellular multiplication of bacteria, it seems likely that bacterial peptides may be presented by the major histocompatibility complex (MHC) class I pathway. To examine this hypothesis, we infected HLA-B*2705- and/or human beta2-microglobulin-transfected murine L-cell lines with M90T, an invasive strain of S. flexneri. Bacterial infection induced no detectable modifications in the biosynthesis and expression level of HLA-B27, as assessed by immunoprecipitation, Northern blot analysis, and flow cytometry. Using confocal microscopy, we observed that bacterial infection induced a clustering of HLA-B27 molecules during macropinocytosis and before bacterial dissemination from cell to cell. Peptides naturally bound to HLA-B27 molecules were acid eluted from infected cells and separated by high-performance liquid chromatography. Major differences were observed in high-performance liquid chromatography profiles and in the nature of peptides presented following bacterial infection. Although most of the antigens presented were not accessed by Edman degradation, we obtained two sequences partially homologous to bacterial proteins. These peptides lacked the major HLA-B27 peptide anchor (Arg) at position 2, and one had an unusual length of 14 amino acids. These data suggest that alterations in the peptide presentation by HLA-B27 occur during infection, which could be relevant to the pathogenesis of HLA-B27-related arthritis.     

Binding and presentation of peptides derived from melanoma antigens MART-1 and glycoprotein-100 by HLA-A2 subtypes. Implications for peptide-based immunotherapy

Cellular immune responses to melanoma-associated Ags are the focus of ongoing studies aimed at developing immunotherapies for treatment of malignant melanoma. Melanoma predominantly affects Caucasians, a population in whom expression of HLA-A2 is prevalent. Among HLA-A2 subtypes, HLA-A*0201 is widely expressed, and HLA-A*0201-restricted, tumor-reactive CTL responses are well studied. We have observed in a group of melanoma patients an unexpectedly high frequency (approximately 20%) of non-HLA-A*0201 subtypes (*0202, *0204, and *0205), and little is known regarding antimelanoma response profiles in patients expressing such subtypes. We analyzed non-HLA-A*0201 peptide response profiles using HLA-A*0201-restricted epitopes from melanoma Ags MART-1/Melan A and glycoprotein 100. Most of these peptides bound to the majority of subtypes tested with 50% inhibitory concentrations less than 500 nM. Recognition of cells pulsed with different peptides (MART-1(27-35), G9(154), and G9(280) Flu M1(58-66)) and expressing different subtype molecules by HLA-A*0201-restricted CTL was limited to only a subset of non-HLA-A*0201 molecules, and the peptide/subtype complexes recognized varied among the effector populations tested. CTL responses elicited from PBL of patients and healthy donors expressing subtypes HLA-A*0202 and HLA-A*0205 suggested significant differences among HLA-A2 subtype function in the context of melanoma Ag presentation. These observations imply the necessity of subtyping patients considered for peptide-based protocols and highlight the need for further study of melanoma-directed cellular responses among patients expressing non-HLA-A*0201 subtypes.