Delineating the role of the HLA-DR4 "shared epitope" in susceptibility versus resistance to develop arthritis

In humans, HLA-DR alleles sharing amino acids at the third hypervariable region with DRB1*0401(shared epitope) are associated with a predisposition to rheumatoid arthritis, whereas DRB1*0402 is not associated with such a predisposition. Both DRB1*0402 and DRB1*0401 occur in linkage with DQ8 (DQB1*0302). We have previously shown that transgenic (Tg) mice expressing HLA-DRB1*0401 develop collagen-induced arthritis. To delineate the role of "shared epitope" and gene complementation between DR and DQ in arthritis, we generated DRB1*0402, DRB1*0401.DQ8, and DRB1*0402.DQ8 Tg mice lacking endogenous class II molecules, AE(o). DRB1*0402 mice are resistant to develop arthritis. In double-Tg mice, the DRB1*0401 gene contributes to the development of collagen-induced arthritis, whereas DRB1*0402 prevents the disease. Humoral response to type II collagen is not defective in resistant mice, although cellular response to type II collagen is lower in *0402 mice compared with *0401 mice. *0402 mice have lower numbers of T cells in thymus compared with *0401 mice, suggesting that the protective effect could be due to deletion of autoreactive T cells. Additionally, DRB1*0402 mice have a higher number of regulatory T cells and show increased activation-induced cell death, which might contribute toward protection. In DRB1*0401.DQ8 mice, activated CD4(+) T cells express class II genes and can present DR4- and DQ8-restricted peptides in vitro, suggesting a role of class II(+) CD4 T cells locally in the joints. The data suggest that polymorphism in DRB1 genes determines predisposition to develop arthritis by shaping the T cell repertoire in thymus and activating autoreactive or regulatory T cells.     

Point mutations in or near the antigen-binding groove of HLA-DR3 implicate class II-associated invariant chain peptide affinity as a constraint on MHC class II polymorphism

During maturation of MHC II molecules, newly synthesized and assembled complexes of MHC II alphabeta dimers with invariant chain (Ii) are targeted to endosomes, where Ii is proteolyzed, leaving remnant class II-associated Ii peptides (CLIP) in the MHC II peptide binding groove. CLIP must be released, usually with assistance from the endosomal MHC II peptide exchange factor, HLA-DM, before MHC II molecules can bind endosomal peptides. Structural factors that control rates of CLIP release remain poorly understood, although peptide side chain-MHC II specificity pocket interactions and MHC II polymorphism are important. Here we report that mutations betaS11F, betaS13Y, betaQ70R, betaK71E, betaK71N, and betaR74Q, which map to the P4 and P6 pockets of the groove of HLA-DR3 molecules, as well as alphaG20E adjacent to the groove, are associated with elevated CLIP in cells. Most of these mutations increase the resistance of CLIP-DR3 complexes to dissociation by SDS. In vitro, the groove mutations increase the stability of CLIP-DR3 complexes to dissociation. Dissociation rates in the presence of DM, as well as coimmunoprecipitation of some mutant DR3 molecules with DM, are also diminished. The profound phenotypes associated with some of these point mutations suggest that the need to maintain efficient CLIP release represents a constraint on naturally occurring MHC II polymorphism.     

Sex influences on the penetrance of HLA shared-epitope genotypes for rheumatoid arthritis.

The association between rheumatoid arthritis (RA) and HLA DRB1 alleles may arise through linkage disequilibrium with a disease locus or the direct involvement of HLA alleles in RA. In support of the latter possibility, the shared-epitope hypothesis has been postulated, stating that conformationally similar DR beta chains encoded by several DRB1 alleles confer disease susceptibility. To examine these alternative hypotheses of marker-disease association and to investigate gender differences in RA susceptibility, we analyzed the distributions of PCR-based DRB1 genotypes of 309 Caucasian RA patients and 283 Caucasian controls. Initially, the marker-association-segregation chi 2 method was used to evaluate evidence for linkage disequilibrium and the direct involvement of markers DR4 Dw4, DR4 Dw14, and DR1 in RA susceptibility. Additional shared-epitope models that grouped DRB1 alleles into five classes (*0401, *0404/*0102, *0405/*0408/*0101, *1001, and all others) and postulated relationships between genotypes and RA susceptibility were also fitted to observed genotypic distributions by the method of minimal chi 2. For females, a linkage-disequilibrium model provided a good fit to the data, as did a shared-epitope model with RA most penetrant among individuals with the *0401,*0401 genotype. For males, the best model indicated highest RA penetrance among shared-epitope compound heterozygotes. Clinically, male RA patients had more subcutaneous nodules and greater use of slowly acting antirheumatic drugs, while female RA patients had earlier disease onset. This study therefore suggests that sex-related factors influence the RA penetrance associated with DRB1 shared-epitope genotypes and that DRB1 effects on RA prognosis and pathogenesis should be considered separately for men and women.     

T cell selection and differential activation on structurally related HLA-DR4 ligands.

Plasticity of TCR interactions during CD4(+) T cell activation by an MHC-peptide complex accommodates variation in the peptide or MHC contact sites in which recognition of an altered ligand by the T cell can modify the T cell response. To explore the contribution of this form of TCR cross-recognition in the context of T cell selection on disease-associated HLA molecules, we have analyzed the relationship between TCR recognition of the DRB1*0401- and DRB1*0404-encoded HLA class II molecules associated with rheumatoid arthritis. Thymic reaggregation cultures demonstrated that CD4(+) T cells selected on either DRB1*0401 or DRB1*0404 could be subsequently activated by the other MHC molecule. Using HLA tetramer technology we identify hemagglutinin residue 307-319-specific T cells restricted by DRB1*0401, but activated by hemagglutinin residues 307-319, in the context of DRB1*0404. One such clone exhibits an altered cytokine profile upon activation with the alternative MHC ligand. This altered phenotype persists when both class II molecules are present. These findings directly demonstrate that T cells selected on an MHC class II molecule carry the potential for activation on altered self ligands when encountering Ags presented on a related class II molecule. In individuals heterozygous for these alleles the possibility of TCR cross-recognition could lead to an aberrant immune response.     

Interaction of MHC class II molecules with the invariant chain: role of the invariant chain (81-90) region.

Association of the invariant chain (Ii) with MHC class II alpha and beta chains is central for their functionality and involves the Ii CLIP(81-104) region. Ii mutants with an antigenic peptide sequence in place of the CLIP region are shown to form alphabetaIi complexes resistant to dissociation by SDS at 25 degrees C. This reflects class II peptide binding site occupancy, since substitution of the major anchor residue within the antigenic peptide sequence of one of these Ii mutants abolishes its capacity to form SDS-stable heterotrimers. Therefore, CLIP location within Ii is compatible with CLIP access to the class II binding groove. However, in wild-type Ii this access does not lead to a tight association, which seems to be affected by the Ii 81-90 region. This region, together with a region C-terminal of CLIP, is shown to contribute to Ii association with HLA-DR1 molecules. Thus, Ii mutants with non-HLA-DR1 binding sequences in place of the CLIP(87-102) region can still associate with HLA-DR1 molecules and inhibit peptide binding.     

Localization of MHC class II/human cartilage glycoprotein-39 complexes in synovia of rheumatoid arthritis patients using complex-specific monoclonal antibodies

Recently human cartilage gp-39 (HC gp-39) was identified as a candidate autoantigen in rheumatoid arthritis (RA). To further investigate the relevance of this Ag in RA, we have generated a set of five mAbs to a combination epitope of complexes of HC gp-39(263-275) and the RA-associated DR alpha beta 1*0401 HLA class II molecules. FACS studies revealed that these mAb recognize specific complexes on homozygous DR alpha beta 1*0401-positive B lymphoblastoid cells pulsed with HC gp-39(263-275). The best mAb, 12A, was further characterized using a set of irrelevant DR alpha beta 1*0401-binding peptides and truncated/elongated versions of HC gp-39(263-275) itself. The minimal epitope recognized in combination with DR alpha beta 1*0401 was HC gp-39(263-273). Peptides not encompassing HC gp-39(263-273) were not recognized. Three of five mAb were able to inhibit (up to 90%) the response of HC gp-39(263-275)-specific DR alpha beta 1*0401-restricted T cell hybridomas to peptide-pulsed APC or purified complexes. Using mAb 12A, we have been able to identify and localize dendritic cells that present DR alpha beta 1*0401/HC gp-39(263-275) complexes in synovial tissue of DR alpha beta 1*0401-positive RA patients, indicating local presentation of the HC gp-39(263-275) epitope in the inflamed target tissue by professional APC. These data support a role of HC gp-39 in the local autoimmune response that leads to chronic inflammation and joint destruction.     

Formation of two peptide/MHC II isomers is catalyzed differentially by HLA-DM

Major histocompatability class II proteins are transmembrane alphabeta-heterodimers that present peptides to T-cells. MHC II may bind exogenous peptides directly at the cell surface. Alternatively, peptides derived from processing of endosomal protein may bind to MHC II in endosomal compartments. There, HLA-DM catalyzes the formation of peptide/MHC complexes, which are then transported to the cell surface. Here we report evidence that the peptide Ii CLIP 81-104 binds to DR*0404 in two alternate registries, whose dissociation rates, while kinetically indistinguishable at pH 5.3 and 37 degrees C, are kinetically resolved in the presence of HLA-DM. In one registry isomer, CLIP Met 91 is placed in the N-terminal P1 pocket of DR*0404, and peptide dissociation is readily catalyzed by HLA-DM. In a second proposed registry, likely with CLIP Leu 97 in the P1 pocket, the complex is substantially less sensitive to HLA-DM catalysis. Without HLA-DM, or at pH 7, the fraction of each isomer formed in solution is relatively insensitive to the duration of incubation with peptide. However, with HLA-DM, the fraction of the DM-insensitive isomer is dramatically influenced by peptide incubation time. The mechanism of isomer formation appears to be determined by the HLA-DM-modified relative association to the two registries, followed by HLA-DM-catalyzed dissociation of each isomer and rebinding, leading to a final isomer composition determined by these kinetic constants. Intramolecular isomer interconversion does not appear to be involved. The behavior of these complexes may provide a model for peptide editing by DM in endosomes.     

Dissection of the HLA-DR4 peptide repertoire in endocrine epithelial cells: strong influence of invariant chain and HLA-DM expression on the nature of ligands

Class II MHC (MHC II) expression is restricted to professional APCs and thymic epithelium but it also occurs in the epithelial cells of autoimmune organs which are the unique targets of the CD4 autoreactive T cells in endocrine autoimmune diseases. This specificity is presumably conditioned by an epithelium-specific peptide repertoire associated to MHC II at the cell surface. MHC II expression and function is dependent on the action of two main chaperones, invariant chain (Ii) and DM, whose expression is coregulated with MHC II. However, there is limited information about the in vivo expression levels of these molecules and uncoordinated expression has been demonstrated in class II-positive epithelial cells that may influence the MHC-associated peptide repertoires and the outcome of the autoimmune response. We have examined the pool of peptides associated to DR4 molecules expressed by a neuroendocrine epithelial cell and the consequences of Ii and DM coexpression. The RINm5F rat insulinoma cell line was transfected with HLA-DRB1*0401, Ii, and DM molecules in four different combinations: RIN-DR4, -DR4Ii, -DR4DM, and -DR4IiDM. The analysis of the peptide repertoire and the identification of the DR4 naturally processed ligands in each transfected cell were achieved by mass spectrometry. The results demonstrate that 1) the expression of Ii and DM affected the DR4 peptide repertoires by producing important variations in their content and in the origin of peptides; 2) these restrictions affected the stability and sequence of the peptides of each repertoire; and 3) Ii and DM had both independent and coordinate effects on these repertoires.     

The role of HLA-DRB1*04 alleles and their association with HLA-DQB genes in genetic susceptibility to rheumatoid arthritis in Hungarian patients

We investigated the HLA-DRB, and DQB polymorphism and haplotypes in RA subjects of Hungarian origin by PCR typing using sequence-specific primers. Molecular subtyping of HLA-DRB1*04 alleles in RA patients showed strongest association with highest relative risk with DRB1*0404. A significantly decreased frequency of DRB1*0403 was observed in patients compared to controls. A significant number of patients carried DR4 haplotypes on DQB1*0302 (54%) relative to DQB1*0301 which was present on 36% of the haplotypes. When compared to controls, the frequency was higher in the latter allele only. Few unique DRB-DQB haplotypes were observed in Hungarian RA patients. In spite of the fact, that the Hungarian population has been isolated linguistically over centuries, a considerable racial admixture has occurred following immigration and invasions, thus the present study confirms in Hungarian patients with RA, previous findings for RA and HLA in European countries.     

Structural basis for the binding of an immunodominant peptide from myelin basic protein in different registers by two HLA-DR2 proteins

Susceptibility to multiple sclerosis (MS) is associated with certain MHC class II haplotypes, in particular HLA-DR2. Two DR beta chains, DRB1*1501 and DRB5*0101, are co-expressed in the HLA-DR2 haplotype, resulting in the formation of two functional cell surface heterodimers, HLA-DR2a (DRA*0101, DRB5*0101) and HLA-DR2b (DRA*0101, DRB1*1501). Both isotypes can present an immunodominant peptide of myelin basic protein (MBP 84-102) to MBP-specific T cells from MS patients. We have determined the crystal structure of HLA-DR2a complexed with MBP 86-105 to 1.9 A resolution. A comparison of this structure with that of HLA-DR2b complexed with MBP 85-99, reported previously, reveals that the peptide register is shifted by three residues, such that the MBP peptide is bound in strikingly different conformations by the two MHC molecules. This shift in binding register is attributable to a large P1 pocket in DR2a, which accommodates Phe92, in conjunction with a relatively shallow P4 pocket, which is occupied by Ile95. In DR2b, by contrast, the small P1 pocket accommodates Val89, while the deep P4 pocket is filled by Phe92. In both complexes, however, the C-terminal half of the peptide is positioned higher in the binding groove than in other MHC class II/peptide structures. As a result of the register shift, different side-chains of the MBP peptide are displayed for interaction with T cell receptors in the DR2a and DR2b complexes. These results demonstrate that MHC molecules can impose different alignments and conformations on the same bound peptide as a consequence of topological differences in their peptide-binding sites, thereby creating distinct T cell epitopes.     

Editing of the HLA-DR-peptide repertoire by HLA-DM.

Antigenic peptide loading of classical major histocompatibility complex (MHC) class II molecules requires the exchange of the endogenous invariant chain fragment CLIP (class II associated Ii peptide) for peptides derived from antigenic proteins. This process is facilitated by the non-classical MHC class II molecule HLA-DM (DM) which catalyzes the removal of CLIP. Up to now it has been unclear whether DM releases self-peptides other than CLIP and thereby modifies the peptide repertoire presented to T cells. Here we report that DM can release a variety of peptides from HLA-DR molecules. DR molecules isolated from lymphoblastoid cell lines were found to carry a sizeable fraction of self-peptides that are sensitive to the action of DM. The structural basis for this DM sensitivity was elucidated by high-performance size exclusion chromatography and a novel mass spectrometry binding assay. The results demonstrate that the overall kinetic stability of a peptide bound to DR determines its sensitivity to removal by DM. We show that DM removes preferentially those peptides that contain at least one suboptimal side chain at one of their anchor positions or those that are shorter than 11 residues. These findings provide a rationale for the previously described ligand motifs and the minimal length requirements of naturally processed DR-associated self-peptides. Thus, in endosomal compartments, where peptide loading takes place, DM can function as a versatile peptide editor that selects for high-stability MHC class II-peptide complexes by kinetic proofreading before these complexes are presented to T cells.     

Redundancy in antigen-presenting function of the HLA-DR and -DQ molecules in the multiple sclerosis-associated HLA-DR2 haplotype

The three HLA class II alleles of the DR2 haplotype, DRB1*1501, DRB5*0101, and DQB1*0602, are in strong linkage disequilibrium and confer most of the genetic risk to multiple sclerosis. Functional redundancy in Ag presentation by these class II molecules would allow recognition by a single TCR of identical peptides with the different restriction elements, facilitating T cell activation and providing one explanation how a disease-associated HLA haplotype could be linked to a CD4+ T cell-mediated autoimmune disease. Using combinatorial peptide libraries and B cell lines expressing single HLA-DR/DQ molecules, we show that two of five in vivo-expanded and likely disease-relevant, cross-reactive cerebrospinal fluid-infiltrating T cell clones use multiple disease-associated HLA class II molecules as restriction elements. One of these T cell clones recognizes >30 identical foreign and human peptides using all DR and DQ molecules of the multiple sclerosis-associated DR2 haplotype. A T cell signaling machinery tuned for efficient responses to weak ligands together with structural features of the TCR-HLA/peptide complex result in this promiscuous HLA class II restriction.     

CLIP-region mediated interaction of Invariant chain with MHC class I molecules

An association between the MHC class II chaperone molecule Invariant chain (Ii) and MHC class I molecules is known to occur, but the basis of the interaction is undetermined. Evidence is presented here that the CLIP region of Ii is involved in this interaction. A peptide encoding residues 91-99 of CLIP (MRMATPLLM) stabilised multiple MHC class I alleles, with the methionine residue at position 99 having a crucial role in binding to H2-K(b), whereas methionine at position 91 also appeared important in binding to RT1-A(a). Ii can also be detected in the class I MHC peptide loading complex. These data provide an explanation for the association of Ii and MHC class I molecules.     

One NY-ESO-1-derived epitope that promiscuously binds to multiple HLA-DR and HLA-DP4 molecules and stimulates autologous CD4+ T cells from patients with NY-ESO-1-expressing melanoma

NY-ESO-1 is expressed by a broad range of human tumors and is often recognized by Abs in the sera of cancer patients with NY-ESO-1-expressing tumors. The NY-ESO-1 gene also encodes several MHC class I- and class II-restricted tumor epitopes recognized by T lymphocytes. In this study we report one novel pan-MHC class II-restricted peptide sequence, NY-ESO-1 87-111, that is capable of binding to multiple HLA-DR and HLA-DP4 molecules, including HLA-DRB1*0101, 0401, 0701, and 1101 and HLA-DPB1*0401 and 0402 molecules. We also demonstrate that peptide NY-ESO-1 87-111 stimulates Th1-type and Th-2/Th0-type CD4(+) T cells and clones when presented in the context of these HLA-DR and HLA-DP4 molecules. Both bulk CD4(+) T cells and CD4(+) T cell clones were capable of recognizing not only peptide-pulsed APCs, but also autologous dendritic cells, either loaded with the NY-ESO-1 protein or transfected with NY-ESO-1 cDNAs. Using IFN-gamma and IL-5 ELISPOT assays and PBL from patients with NY-ESO-1-expressing tumors, we observed the existence of Th1-type circulating CD4(+) T cells recognizing peptide NY-ESO-1 87-111 in the context of HLA-DP4 molecules. Taken together, these data represent the first report of an HLA-DR- and HLA-DP-restricted epitope from a tumor Ag. They also support the relevance of cancer vaccine trials with peptides NY-ESO-1 87-111 in the large number of cancer patients with NY-ESO-1-expressing tumors.     

Effect of decreasing the affinity of the class II-associated invariant chain peptide on the MHC class II peptide repertoire in the presence or absence of H-2M

The class II-associated invariant chain peptide (CLIP) region of the invariant chain (Ii) directly influences MHC class II presentation by occupying the MHC class II peptide-binding groove, thereby preventing premature loading of peptides. Different MHC class II alleles exhibit distinct affinities for CLIP, and a low affinity interaction has been associated with decreased dependence upon H-2M and increased susceptibility to rheumatoid arthritis, suggesting that decreased CLIP affinity alters the MHC class II-bound peptide repertoire, thereby promoting autoimmunity. To examine the role of CLIP affinity in determining the MHC class II peptide repertoire, we generated transgenic mice expressing either wild-type human Ii or human Ii containing a CLIP region of low affinity for MHC class II. Our data indicate that although degradation intermediates of Ii containing a CLIP region with decreased affinity for MHC class II do not remain associated with I-A(b), this does not substantially alter the peptide repertoire bound by MHC class II or increase autoimmune susceptibility in the mice. This implies that the affinity of the CLIP:MHC class II interaction is not a strong contributory factor in determining the probability of developing autoimmunity. In contrast, in the absence of H-2M, MHC class II peptide repertoire diversity is enhanced by decreasing the affinity of CLIP for MHC class II, although MHC class II cell surface expression is reduced. Thus, we show clearly, in vivo, the critical chaperone function of H-2M, which preserves MHC class II molecules for high affinity peptide binding upon dissociation of Ii degradation intermediates.     

Humanized mice as a model for rheumatoid arthritis

Genetic susceptibility to rheumatoid arthritis (RA), a common autoimmune disease, is associated with certain HLA-DR4 alleles. Treatments are rarely curative and are often tied to major side effects. We describe the development of a humanized mouse model wherein new, less toxic, vaccine-like treatments for RA might be pretested. This model includes four separate transgenes: HLA-DR*0401 and human CD4 molecules, a RA-related human autoantigenic protein (HCgp-39), and a T-cell receptor (TCRalphabeta) transgene specific for an important HCgp-39 epitope, eliciting strong Th1 responses in the context of HLA-DR*0401.     

HLA-DR1 (DRB1*0101) and DR4 (DRB1*0401) use the same anchor residues for binding an immunodominant peptide derived from human type II collagen.

Rheumatoid arthritis is an autoimmune disease in which susceptibility is strongly associated with the expression of specific HLA-DR haplotypes, including DR1 (DRB1*0101) and DR4 (DRB1*0401). As transgenes, both of these class II molecules mediate susceptibility to an autoimmune arthritis induced by immunization with human type II collagen (hCII). The dominant T cell response of both the DR1 and DR4 transgenic mice to hCII is focused on the same determinant core, CII(263-270). Peptide binding studies revealed that the affinity of DR1 and DR4 for CII(263-270) was at least 10 times less than that of the model Ag HA(307-319), and that the affinity of DR4 for the CII peptide is 3-fold less than that of DR1. As predicted based on the crystal structures, the majority of the CII-peptide binding affinity for DR1 and DR4 is controlled by the Phe(263); however, unexpectedly the adjacent Lys(264) also contributed significantly to the binding affinity of the peptide. Only these two CII amino acids were found to provide binding anchors. Amino acid substitutions at the remaining positions had either no effect or significantly increased the affinity of the hCII peptide. Affinity-enhancing substitutions frequently involved replacement of a negative charge, or Gly or Pro, hallmark amino acids of CII structure. These data indicate that DR1 and DR4 bind this CII peptide in a nearly identical manner and that the primary structure of CII may dictate a different binding motif for DR1 and DR4 than has been described for other peptides that bind to these alleles.     

HLA-DR4 molecules in neuroendocrine epithelial cells associate to a heterogeneous repertoire of cytoplasmic and surface self peptides

Expression of MHC class II genes by epithelial cells is induced in inflammatory conditions such as autoimmunity and organ transplantation. Class II ligands generated by the epithelial cell processing mechanisms are unknown, although some unique epitopes have been described in epithelial cells that B cells could not generate. Epithelial cells are the targets of autoreactive T cell responses in autoimmune diseases and of transplant rejection processes, which may involve recognition of cell type-specific epitopes. In the present report, we have compared the DR4-associated repertoire and the intracellular distribution of class II, invariant chain (Ii), and DM molecules between a human DR4-, Ii-, and DM-transfected rat neuroendocrine epithelial cell line and a homozygous DR4 (DRB1*0401) lymphoblastoid B cell line, by mass spectrometry sequencing techniques, and immunoelectron microscopy. The epithelial cells chosen for transfection, RINm5F, are rat insular cells widely used for human studies of autoimmune diabetes. The results revealed a remarkably heterogeneous pool of self protein-derived peptides from the cell surface and various intracellular compartments, including the cytosol and secretory vesicles in epithelial cells, compared with a very restricted homogeneous repertoire in lymphoblastoid B cell lines, where few epitopes from surface molecules were predominant. The generation of distinct DR4-associated peptide repertoires in these two cell types could be due to the effect of several factors including differences in subcellular location of Ii and DM molecules, differential DO expression, and cell type-specific mechanisms of class II ligand generation. This is specially relevant to processes involving epithelial T cell interactions such as organ-specific autoimmunity and transplant rejection.     

Autoantigenic HCgp39 epitopes are presented by the HLA-DM-dependent presentation pathway in human B cells

It is hypothesized that autoimmune diseases manifest when tolerance to self-Ags fails. One possible mechanism to break tolerance is presentation of self-Ag in an altered form. Most Ags are presented by APCs via the traditional presentation pathway that includes "epitope editing" by intracellular HLA-DM, a molecule that selects for stable MHC-peptide complexes. We were interested in testing the hypothesis that autoreactive MHC-peptide complexes may reach the cell surface by an alternate pathway without being edited by HLA-DM. We selected a cartilage autoantigen human cartilage glycoprotein 39 to which T cell responses are observed in rheumatoid arthritis (RA) patients and some DR(*)04 healthy subjects. RA is genetically associated with certain DRB1 alleles, including DRB1(*)0401 but closely related allele DRB1(*)0402 is either neutral or mildly protective with respect to RA. We generated human B lymphoblastoid cell line cells expressing DR(*)0401 or DR(*)0402 in the presence or absence of intracellular HLA-DM and assessed their ability to present a candidate autoantigen, human cartilage glycoprotein 39. Our results show that the presence of intracellular HLA-DM is critical for presentation of this autoantigen to CD4(+) T cell hybridomas generated from DR(*)04-transgenic mice. Presentation of an autoantigen by the traditional HLA-DM-dependent pathway has implications for Ag presentation events in RA.     

An HLA-DRB1-derived peptide associated with protection against rheumatoid arthritis is naturally processed by human APCs

Predisposition to rheumatoid arthritis (RA) is thought to be associated with HLA-DR1, -DR4, and -DR10. However, many epidemiological observations are better explained by a model in which the DQ alleles that are linked to these DR alleles, i.e., DQ5, DQ7, and DQ8, predispose to RA, while certain DR alleles have a dominant protective effect. All protective DRB1 alleles, e.g., *0402, *1301, and *1302, encode a unique motif, (70)DERAA(74). The protection may be explained by the presentation of DRB1-derived peptides by DQ to immunoregulatory T cells, because it was demonstrated in various autoimmune disease models that T cell responses to certain self-Ags can be involved in disease suppression. The aim of this study was to analyze whether peptides carrying the DERAA motif are naturally processed by human APC and presented in the context of the RA-predisposing DQ. Using a synthetic peptide carrying the DRB1*0402-derived sequence (65)KDILEDERAAVDTYC(79), we generated DERAA peptide-specific DQ-restricted T cell clones (TCC) from a DQ8 homozygous individual carrying DERAA-negative DR4 alleles. By analyzing the proliferation of these TCC, we demonstrated natural processing and presentation of the DERAA sequence by the APC of all the individuals (n = 12) carrying a DERAA-positive DRB1 allele and either DQ8 or the DQ8-related DQ7. Using a panel of truncated synthetic peptides, we identified the sequence (67)(I)LEDERAAVD(TY)(78) as the minimal determinant for binding to DQ8 and for recognition by the TCC. These findings support a model in which self-MHC-derived peptide can modulate predisposition to autoimmune disease in humans.