Ligandomes obtained from different HLA-class II-molecules are homologous for N- and C-terminal residues outside the peptide-binding cleft.

Human CD4+ T lymphocytes play an important role in inducing potent immune responses. T cells are activated and stimulated by peptides presented in human leucocyte antigen (HLA)-class II molecules. These HLA-class II molecules typically present peptides of between 12 and 20 amino acids in length. The region that interacts with the HLA molecule, designated as the peptide-binding core, is highly conserved in the residues which anchor the peptide to the molecule. In addition, as these peptides are the product of proteolytic cleavages, certain conserved residues may be expected at the N- and C-termini outside the binding core. To study whether similar conserved residues are present in different cell types, potentially harbouring different proteolytic enzymes, the ligandomes of HLA-DRB1*03:01/HLA-DRB > 1 derived from two different cell types (dendritic cells and EBV-transformed B cells) were identified with mass spectrometry and the binding core and N- and C-terminal residues of a total of 16,568 peptides were analysed using the frequencies of the amino acids in the human proteome. Similar binding motifs were found as well as comparable conservations in the N- and C-terminal residues. Furthermore, the terminal conservations of these ligandomes were compared to the N- and C-terminal conservations of the ligandome acquired from dendritic cells homozygous for HLA-DRB1*04:01. Again, comparable conservations were evident with only minor differences. Taken together, these data show that there are conservations in the terminal residues of peptides, presumably the result of the activity of proteases involved in antigen processing.

The increased ability to present citrullinated peptides is not unique to HLA-SE molecules: arginine-to-citrulline conversion also enhances peptide affinity for HLA-DQ molecules.

BACKGROUND: Presentation of citrullinated neo-epitopes by HLA-DRB1 molecules that carry the shared epitope (SE) sequence was proposed to explain the association between HLA and seropositive RA. Although it is shown that several HLA-DRB1-SE molecules display enhanced binding affinities for citrullinated ligands, the ability of other HLA molecules to present citrullinated epitopes has not been investigated in a systematic manner. To better understand the HLA-RA connection, we aimed to investigate if the enhanced capacity to present arginine-to-citrulline-converted peptides is unique for HLA-SE alleles. METHODS: We selected four HLA molecules (one HLA-DR and three HLA-DQ molecules) that could potentially prefer citrulline over arginine residues in specific pockets and in addition two HLA-SE alleles as a method validation control. The affinity of peptides containing arginine/citrulline residues at positions interacting with the various peptide-binding pockets was compared by HLA class II peptide affinity assays. RESULTS: Pocket 4 of HLA-DRB1*04:04 and -DRB1*04:05 displayed a preference for citrulline over arginine, a property found in other pockets as well. HLA-DRB1*03:01 did not display an enhanced affinity for peptides containing a citrulline. In contrast, several peptide-binding pockets of the analyzed HLA-DQ molecules showed enhanced affinities for citrulline compared to arginine residues: i.e., pockets 4, 6, 7, and 9 of HLA-DQ2 and pockets 1, 6, and 9 of HLA-DQ7 and HLA-DQ8. CONCLUSIONS: Arginine-to-citrulline conversion of peptides can also enhance the binding affinity for non-HLA-SE molecules. Hence the capacity to present citrullinated neo-epitopes is not confined to HLA-SE molecules, opening the possibility that also other HLA molecules could potentiate a possible breach of T cell tolerance toward citrullinated antigens.

Protective effect of HLA-DRB1*13 alleles during specific phases in the development of ACPA-positive RA.

OBJECTIVE: Human leucocyte antigen (HLA)-DRB1*13 alleles are associated with protection from anticitrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA). It is, however, unknown at which phase of disease development (seroconversion, ACPA maturation, disease onset or outcome) these alleles are most important. We therefore examined the effect of HLA-DRB1*13 on: ACPA presence (systemic autoimmunity associated with RA) in individuals with and without RA, on ACPA characteristics and on clinical outcome measures. METHODS: The effect of HLA-DRB1*13 on ACPA presence in subjects with or without RA (non-RA) was assessed in the Swedish twin registry (n=10 748). ACPA characteristics were studied in patients with ACPA-positive RA from the Swedish Epidemiological Investigation of RA (EIRA, n=1224) and the Dutch Leiden Early Arthritis Clinic (EAC, n=441). Disease activity at inclusion and disease outcome (disease-modifying antirheumatic drugs (DMARD)-free sustained remission and radiographic progression) was assessed in patients with RA from the EAC. RESULTS: HLA-DRB1*13 is associated with protection from ACPA-positive RA (prevalence 16% vs 28% in ACPA-negative non-RA), but not with significant protection from ACPA in individuals without RA (prevalence: 22%, p value 0.09). HLA-DRB1*13 is associated with lower ACPA-levels (EIRA: 447 U/ml versus 691 U/ml, p value= 0.0002) and decreased citrullinated epitope recognition (EIRA: p<0.0001). No association between HLA-DRB1*13 and disease activity or outcome was found. CONCLUSIONS: These data indicate that HLA-DRB1*13 mainly affects the onset of ACPA-positive RA in ACPA-positive non-RA individuals. In RA, HLA-DRB1*13 influences ACPA characteristics but not the disease course. This implies that therapeutic strategies aimed at emulating the HLA-DBR1*13 protective effect may be most effective in ACPA-positive healthy individuals at risk for RA.

A new classification of HLA-DRB1 alleles based on acid-base properties of the amino acids located at positions 13, 70 and 71: impact on ACPA status or structural progression, and meta-analysis on 1235 patients with rheumatoid from two cohorts (ESPOIR and EAC cohort).

OBJECTIVE: To group HLA-DRB1 alleles based on acid-base properties of amino acids at positions 13, 70 and 71 and analyse their association with the presence of anticitrullinated peptide antibodies (ACPA) and structural progression in 2 cohorts of early rheumatoid arthritis (RA). METHODS: Patients with RA (N=612) from ESPOIR cohort and from EAC cohort (n=624) were genotyped for HLA-DRB1 alleles. The alleles containing the RAA sequence at positions 72-74 were classified into 3 groups according to the amino acid at positions 13, 70 and 71: BB encoding basic amino acids at positions 13, 70 and 71; A encoding acidic amino acids at positions 70 and 71; and BN encoding either neutral amino acids at position 13 and basic amino acids at positions 70 and 71, or basic amino acid at position 13 and neutral amino acids at positions 70 and 71. The associations between the different alleles and (1) the ACPA presence, and (2) the structural progression were assessed by χ(2) test; a meta-analysis was performed on the 2 cohorts using the Mantel-Haenszel method. RESULTS: After meta-analysis, BB alleles were significantly associated with ACPA presence (OR (95% CI) 4.08 (3.14 to 5.31)) and structural progression (OR (95% CI) 2.33 (1.76 to 3.09)). The alleles protected significantly against ACPA presence (OR (95% CI) 0.37 (0.28 to 0.50)) and structural progression (OR (95% CI) 0.34 (0.23 to 0.50)). This acid-base classification allowed to separate another group BN with an intermediate risk of ACPA production (OR (95% CI) 1.14 (0.91 to 1.44)) and structural progression (OR (95% CI) 1.01 (0.77 to 1.33)). CONCLUSIONS: This new classification permitted to make a hierarchy of HLA-DRB1 alleles in terms of association with ACPA presence or structural progression in early RA.

An investigation of the added value of an ACPA multiplex assay in an early rheumatoid arthritis setting.

INTRODUCTION: Recently, arrays have become available that allow the simultaneous analysis of several anti-citrullinated protein antibody (ACPA) reactivities using distinct citrullinated peptides. Such assays are designed for exploratory studies. The interpretation of positive antibody reactivities can best be made if the diagnostic and prognostic value of a multiplex array in an early arthritis setting is known and if the multiplex-positive patients who are negative according to three commonly used commercial ACPA assays are characterized. METHODS: Using Thermo Scientific's ImmunoCap ISAC (Immuno Solid-phase Allergen Chip) system, a multiplexed array that determines reactivities to 11 citrullinated peptides, we analysed serum/plasma of 195 healthy controls and 1282 early arthritis patients from two independent cohorts: the Leiden Early Arthritis Clinic (n = 1013) and the IMPROVED (n = 269) cohort. Findings were compared with results primarily of the anti-citrullinated cyclic peptide 2 (anti-CCP-2) assay but also with anti- CCP-3 and anti-mutated citrullinated vimentin (anti-MCV) assays. The associations between ACPA reactivities and patient characteristics, risk factors (shared epitope, smoking) and disease outcomes (progression of undifferentiated arthritis to rheumatoid arthritis (RA) and severity of joint destruction) were assessed. RESULTS: Thirty-one percent of anti-CCP-2-negative RA patients displayed reactivity toward citrullinated peptides in the multiplex assay. These patients had a positive signal toward a more restricted peptide repertoire than anti-CCP-2-positive RA patients (median of 1 versus 5). Within anti-CCP-2-negative patients, ACPA reactivity as detected by multiplex array was not significantly associated with known risk factors or clinical or prognostic parameters. The frequency of sera from anti-CCP-2-negative RA patients who were positive for the multiplexed peptides was comparable to the frequency in non-RA arthritic patients (27 %). CONCLUSIONS: Additive citrulline peptide reactivities detected by the current multiplex system did not reach significant power to be RA-specific. The presence of residual citrulline reactivities detected by this multiplex system in arthritis patients who are negative in commercial ACPA assays needs to be interpreted with caution.

Crossreactivity to vinculin and microbes provides a molecular basis for HLA-based protection against rheumatoid arthritis.

The HLA locus is the strongest risk factor for anti-citrullinated protein antibody (ACPA)(+) rheumatoid arthritis (RA). Despite considerable efforts in the last 35 years, this association is poorly understood. Here we identify (citrullinated) vinculin, present in the joints of ACPA(+) RA patients, as an autoantigen targeted by ACPA and CD4(+) T cells. These T cells recognize an epitope with the core sequence DERAA, which is also found in many microbes and in protective HLA-DRB1*13 molecules, presented by predisposing HLA-DQ molecules. Moreover, these T cells crossreact with vinculin-derived and microbial-derived DERAA epitopes. Intriguingly, DERAA-directed T cells are not detected in HLA-DRB1*13(+) donors, indicating that the DERAA epitope from HLA-DRB1*13 mediates (thymic) tolerance in these donors and explaining the protective effects associated with HLA-DRB1*13. Together our data indicate the involvement of pathogen-induced DERAA-directed T cells in the HLA-RA association and provide a molecular basis for the contribution of protective/predisposing HLA alleles.

Fine-mapping the human leukocyte antigen locus in rheumatoid arthritis and other rheumatic diseases: identifying causal amino acid variants?

PURPOSE OF REVIEW: To provide an update on and the context of the recent findings obtained with novel statistical methods on the association of the human leukocyte antigen (HLA) locus with rheumatic diseases. RECENT FINDINGS: Novel single nucleotide polymorphism fine-mapping data obtained for the HLA locus have indicated the strongest association with amino acid positions 11 and 13 of HLA-DRB1 molecule for several rheumatic diseases. On the basis of these data, a dominant role for position 11/13 in driving the association with these diseases is proposed and the identification of causal variants in the HLA region in relation to disease susceptibility implicated. SUMMARY: The HLA class II locus is the most important risk factor for several rheumatic diseases. Recently, new statistical approaches have identified previously unrecognized amino acid positions in the HLA-DR molecule that associate with anticitrullinated protein antibody-negative and anticitrullinated protein antibody-positive rheumatoid arthritis. Likewise, similar findings have been made for other rheumatic conditions such as giant-cell arteritis and systemic lupus erythematosus. Interestingly, all these studies point toward an association with the same amino acid positions: amino acid positions 11 and 13 of the HLA-DR ß chain. As both these positions influence peptide binding by HLA-DR and have been implicated in antigen presentation, the novel fine-mapping approach is proposed to map causal variants in the HLA region relevant to rheumatoid arthritis and several rheumatic diseases. If these interpretations are correct, they would direct the biological research aiming to address the explanation for the HLA-disease association. Here, we provide an overview of the recent findings and evidence from literature that, although relevant new insights have been obtained on HLA-disease associations, the interpretation of the biological role of these amino acids as causal variants explaining that such associations should be taken with caution.

HLA and rheumatoid arthritis: how do they connect?

Rheumatoid arthritis (RA) is a destructive autoimmune disease that mainly affects synovial joints. RA patients can be subdivided in two distinct disease subsets based on the presence of anti-citrullinated protein antibodies (ACPA). These two disease phenotypes are associated with different environmental and genetic risk factors and clinical parameters. The HLA class II locus is the most important risk factor for ACPA-positive RA (ACPA+ RA). ACPA can be found up to 10 years before diagnosis and can be used as a predictive biomarker. During progression from breaking tolerance to a citrullinated protein to ACPA+ RA, the ACPA response matures. Recent work implicates the HLA class II locus as a risk factor in the progression from ACPA positivity to ACPA+ RA. We now propose that this locus directly influences the maturation of the ACPA response, most likely via antigen-specific T-cells providing help to ACPA-producing B-cells allowing for maturation of the citrullinated protein-specific autoantibody response. We present and discuss several models and underlying data, including antibody cross-reactivity, molecular mimicry, and neo-antigen formation, that could explain the HLA-RA connection.

A molecular basis for the association of the HLA-DRB1 locus, citrullination, and rheumatoid arthritis.

Rheumatoid arthritis (RA) is strongly associated with the human leukocyte antigen (HLA)-DRB1 locus that possesses the shared susceptibility epitope (SE) and the citrullination of self-antigens. We show how citrullinated aggrecan and vimentin epitopes bind to HLA-DRB1*04:01/04. Citrulline was accommodated within the electropositive P4 pocket of HLA-DRB1*04:01/04, whereas the electronegative P4 pocket of the RA-resistant HLA-DRB1*04:02 allomorph interacted with arginine or citrulline-containing epitopes. Peptide elution studies revealed P4 arginine-containing peptides from HLA-DRB1*04:02, but not from HLA-DRB1*04:01/04. Citrullination altered protease susceptibility of vimentin, thereby generating self-epitopes that are presented to T cells in HLA-DRB1*04:01(+) individuals. Using HLA-II tetramers, we observed citrullinated vimentin- and aggrecan-specific CD4(+) T cells in the peripheral blood of HLA-DRB1*04:01(+) RA-affected and healthy individuals. In RA patients, autoreactive T cell numbers correlated with disease activity and were deficient in regulatory T cells relative to healthy individuals. These findings reshape our understanding of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in RA.

Communication between human mast cells and CD4(+) T cells through antigen-dependent interactions.

Mast cells (MCs) are immune cells residing in tissues where pathogens are first encountered. It has been indicated that MCs might also be involved in setting the outcome of T-cell responses. However, little is known about the capacity of human MCs to express MHC class II and/or to capture and present antigens to CD4(+) T cells. To study the T-cell stimulatory potential of human MCs, CD34(+) stem cell derived MCs were generated. These cells expressed HLA-DR when stimulated with IFN-γ, and, importantly, presented peptide and protein for activation of antigen-specific CD4(+) T cells. The interplay between MC and T cell led to increased HLA-DR expression on MCs. MCs were present in close proximity to T cells in tonsil and expressed HLA-DR and CD80, indicating their ability to present antigens to CD4(+) T cells in T-cell areas of human LNs. Our data show that MCs can present native antigens to human CD4(+) T cells and that HLA-DR expressing MCs are present in tonsil tissue, indicating that human MCs can directly activate T cells and provide a rationale to study the potential of MCs to prime and/or skew human T-cell responses.

Genetics of rheumatoid arthritis: what have we learned?

Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting 0.5-1% of the population worldwide. The disease has a heterogeneous character, including clinical subsets of anti-citrullinated protein antibody (ACPA)-positive and APCA-negative disease. Although the pathogenesis of RA is poorly understood, progress has been made in identifying genetic factors that contribute to the disease. The most important genetic risk factor for RA is found in the human leukocyte antigen (HLA) locus. In particular, the HLA molecules carrying the amino acid sequence QKRAA, QRRAA, or RRRAA at positions 70-74 of the DRß1 chain are associated with the disease. The HLA molecules carrying these "shared epitope" sequences only predispose for ACPA-positive disease. More than two decades after the discovery of HLA-DRB1 as a genetic risk factor, the second genetic risk factor for RA was identified in 2003. The introduction of new techniques, such as methods to perform genome-wide association has led to the identification of more than 20 additional genetic risk factors within the last 4 years, with most of these factors being located near genes implicated in immunological pathways. These findings underscore the role of the immune system in RA pathogenesis and may provide valuable insight into the specific pathways that cause RA.