HLA-B*08 Identified as the Most Prominently Associated Major Histocompatibility Complex Locus for Anti-Carbamylated Protein Antibody-Positive/Anti-Cyclic Citrullinated Peptide-Negative Rheumatoid Arthritis.

OBJECTIVE: Previously, only the HLA-DRB1 alleles have been assessed in rheumatoid arthritis (RA). The aim of the present study was to identify the key major histocompatibility complex (MHC) susceptibility factors showing a significant association with anti-carbamylated protein antibody-positive (anti-CarP+) RA. METHODS: Analyses were restricted to RA patients who were anti-cyclic citrullinated peptide antibody negative (anti-CCP-), because the anti-CCP status dominated the results otherwise. Therefore, we studied samples from 1,821 anti-CCP- RA patients and 6,821 population controls from Spain, Sweden, and the Netherlands. The genotypes for ~8,000 MHC biallelic variants were assessed by dense genotyping and imputation. Their association with the anti-CarP status in RA patients was tested with logistic regression and combined with inverse-variance meta-analysis. Significance of the associations was assessed according to a study-specific threshold of P < 2.0 × 10-5 . RESULTS: The HLA-B*08 allele and its correlated amino acid variant Asp-9 showed a significant association with anti-CarP+/anti-CCP- RA (P < 3.78 × 10-7 ; I2 = 0). This association was specific when assessed relative to 3 comparator groups: population controls, anti-CarP-/anti-CCP- RA patients, and anti-CCP- RA patients who were positive for other anti-citrullinated protein antibodies. Based on these findings, anti-CarP+/anti-CCP- RA patients could be separated from other antibody-defined subsets of RA patients in whom an association with the HLA-B*08 allele has been previously demonstrated. No other MHC variant remained associated with anti-CarP+/anti-CCP- RA after accounting for the presence of the HLA-B*08 allele. Specifically, the reported association of HLA-DRB1*03 was observed at a level comparable to that reported previously, but it was attributable to linkage disequilibrium. CONCLUSION: These results identify HLA-B*08 carrying Asp-9 as the MHC locus showing the strongest association with anti-CarP+/anti-CCP- RA. This knowledge may help clarify the role of the HLA in susceptibility to specific subsets of RA, by shaping the spectrum of RA autoantibodies.

Oral Lichen Planus - Related Connection with HLA-System Antigens.

AIM: To determine whether there is an immunogenic connection and antigen difference between the HLA antigens in the erosive (EOLP) and reticular (ROLP) oral lichen planus. MATERIALS AND METHOD: 73 patients with ROLP and EOLP have been tested. Typing of the HLA antigens has been made for locus A and B. The typing of the HLA was conducted with the use of microlymphocyto toxic test by Terasaki. The reading of the findings has been conducted with an inverse microscope. When a reaction has 4 points it is considered to be positive. RESULTS: The most frequently typified antigens in ROLP from locus A are HLA А2 (57.57%) and А3 (33.33)%, and for locus B 21.21%. In EOLP it is А9 (8888%). In locus B a connection has been found with HLA B8 (77.77%). The statistical analysis with the ×2 test has shown that the carriers of HLA A9 display a relative risk (RR) of 3.65 and ×2=20.72. Consequently, there is high static importance for locus A p<0,001. For locus B, In EOLP for HLA B8, RR=6. 7 ×2=37.64 and p<0,001. ROLP has shown association with HLA A3, where RR=2. 31 and ×2 =9.14 and p<0.05. CONCLUSIONS: In ROLP A3 antigen and in EOLP A9 and A8 may be considered as carriers with proneness to OLP.

Platelets from donors with consistently low HLA-B8, -B12, or -B35 expression do not undergo antibody-mediated internalization.

Patients refractory to platelet transfusions because of alloimmunization require HLA-matched platelets, which is only possible if a large HLA-typed donor pool is available. However, even then, patients with broad immunization or rare haplotypes may not have suitable donors. In these patients, transfusions with platelets showing low HLA class I expression may be an alternative to fully HLA-matched transfusions. In this study, we quantified the proportion of donors with consistently low HLA-B8, -B12, and -B35 expression on platelets using human monoclonal antibodies specific for these antigens. Furthermore, as model for in vivo clearance, antibody-mediated internalization of these platelets by macrophages was investigated. The expression of HLA-B8, -B12, or -B35 on platelets was extremely variable between individuals (coefficients of variation, 41.4% to 73.6%). For HLA-B8, but not for HLA-B12 or -B35, this variation was in part explained by zygosity. The variation was most pronounced in, but not exclusive to, platelets. Expression within one donor was consistent over time. Remarkably, 32% of 113 HLA-B8, 34% of 98 HLA-B12, and 9% of 66 HLA-B35 donors showed platelet antigen expression that was not or only minimally above background. Antibody-mediated internalization of platelets by macrophages correlated with antibody opsonization and antigen expression and was absent in platelets with low or minimal HLA expression. In conclusion, our findings indicate that a substantial proportion of donors have platelets with consistently low expression of specific HLA class I antigens. These platelets may be used to treat refractory patients with antibodies directed against these particular antigens, despite HLA mismatches.

The novel HLA-B*08:183 allele identified by sequence-based typing in a Caucasian leukemia patient.

HLA-B*08:183 differs from HLA-B*08:01:01 by a single substitution in exon 5.

Gliadin-Specific CD8+ T Cell Responses Restricted by HLA Class I A*0101 and B*0801 Molecules in Celiac Disease Patients.

Initial studies associated the HLA class I A*01 and B*08 alleles with celiac disease (CD) susceptibility. Subsequent analyses showed a primary association with HLA class II alleles encoding for the HLA DQ2.5 molecule. Because of the strong linkage disequilibrium of A*01 and B*08 alleles with the DR3-DQ2.5 haplotype and a recent genome-wide association study indicating that B*08 and B*39 are predisposing genes, the etiologic role of HLA class I in CD pathogenesis needs to be addressed. We screened gliadin proteins (2α-, 2ω-, and 2γ-gliadin) using bioinformatic algorithms for the presence of peptides predicted to bind A*0101 and B*0801 molecules. The top 1% scoring 9- and 10-mer peptides (N = 97, total) were synthesized and tested in binding assays using purified A*0101 and B*0801 molecules. Twenty of ninety-seven peptides bound B*0801 and only 3 of 97 bound A*0101 with high affinity (IC50 < 500 nM). These 23 gliadin peptides were next assayed by IFN-γ ELISPOT for recognition in peripheral blood cells of CD patients and healthy controls carrying the A*0101 and/or B*0801 genes and in A*0101/B*0801- CD patients. Ten of the twenty-three peptides assayed recalled IFN-γ responses mediated by CD8+ T cells in A*0101/B*0801+ patients with CD. Two peptides were restricted by A*0101, and eight were restricted by B*0801. Of note, 50% (5/10) of CD8+ T cell epitopes mapped within the γ-gliadins. Our results highlight the value of predicted binding to HLA molecules for identifying gliadin epitopes and demonstrate that HLA class I molecules restrict the anti-gluten T cell response in CD patients.

Clinical and genetic associations of radiographic sacroiliitis and its different patterns in psoriatic arthritis.

OBJECTIVES: We aimed to 1) identify clinical and genetic associations of sacroiliitis (SI) in patients with psoriatic arthritis (PsA), and 2) describe the different radiographic patterns of SI in PsA and their clinical and genetic associations. METHODS: 283 PsA patients, fulfilling CASPAR criteria, underwent detailed skin and rheumatologic assessments. In addition, HLA-B*27 and B*080101 status was recorded, which have been shown as the key genetic markers of radiographic SI in PsA. Grade 2 Unilateral or bilateral radiographic changes of SI were required for inclusion and involvement was further defined as asymmetrical or symmetrical. RESULTS: 70 patients (25%) had radiographic SI; all either with a present or past history of backache. Regression analysis demonstrated a significant association of SI with peripheral joint erosions (p=0.043), PASI maximum (p=0.041), younger age of PsA onset (p=<0.001), presence of HLA-B*0801 (p=0.002) and only marginal significance with HLA-B*2705 (p=0.059). Asymmetrical SI was noted in 51 patients (73%). In striking contrast to those patients with symmetrical SI, patients with asymmetrical SI were more likely to be female (p=0.04), have a trend towards more severe nail disease (p=0.08) and peripheral joint erosions (p=0.08), more osteolysis (p=0.01), more HLA-B*0801 positivity (p=0.001) and much less HLA-B*270502 positivity (p=<0.001). CONCLUSIONS: PsA developing at a younger age, severe skin disease, peripheral joint erosions, and HLA-B*0801 are significantly associated with SI, and there was only a marginal trend towards significance for HLA-B*2705. HLA-B*27 positive Axial-PsA patients resemble AS, while HLA-B*0801 positive Axial-PsA patients have asymmetrical and/or unilateral SI, which are typical of PsA.

Identification of 3 novel HLA-B alleles: B*08:173, B*18:72:03 and B*53:05:02.

A total of 3 novel human leukocyte antigen-B (HLA-B) alleles were detected by next generation sequencing and confirmed by monoallelic sequencing.

Identification of a novel HLA allele, HLA-B*08:163, in a platelet donor.

The novel HLA-B*08:163 allele differs from HLA-B*08:01:01:01 by a single nucleotide substitution at codon 105.

Cell surface expression level variation between two common Human Leukocyte Antigen alleles, HLA-A2 and HLA-B8, is dependent on the structure of the C terminal part of the alpha 2 and the alpha 3 domains.

Constitutive cell surface expression of Human Leukocyte Antigen (HLA) class I antigens vary extremely from tissue to tissue and individual antigens may differ widely in expression levels. Down-regulation of class I expression is a known immune evasive mechanism used by cancer cells and viruses. Moreover, recent observations suggest that even minor differences in expression levels may influence the course of viral infections and the frequency of complications to stem cell transplantation. We have shown that some human multipotent stem cells have high expression of HLA-A while HLA-B is only weakly expressed, and demonstrate here that this is also the case for the human embryonic kidney cell line HEK293T. Using quantitative flow cytometry and quantitative polymerase chain reaction we found expression levels of endogenous HLA-A3 (median 71,204 molecules per cell) 9.2-fold higher than the expression of-B7 (P = 0.002). Transfection experiments with full-length HLA-A2 and -B8 encoding plasmids confirmed this (54,031 molecules per cell vs. 2,466, respectively, P = 0.001) independently of transcript levels suggesting a post-transcriptional regulation. Using chimeric constructs we found that the cytoplasmic tail and the transmembrane region had no impact on the differential cell surface expression. In contrast, ~65% of the difference could be mapped to the six C-terminal amino acids of the alpha 2 domain and the alpha 3 domain (amino acids 176-284), i.e. amino acids not previously shown to be of importance for differential expression levels of HLA class I molecules. We suggest that the differential cell surface expression of two common HLA-A and-B alleles is regulated by a post-translational mechanism that may involve hitherto unrecognized molecules.

Design and validation of conditional ligands for HLA-B*08:01, HLA-B*15:01, HLA-B*35:01, and HLA-B*44:05.

We designed conditional ligands restricted to HLA-B*08:01, -B*35:01, and -B*44:05 and proved the use of a conditional ligand previously designed for HLA-B*15:02 together with HLA-B*15:01. Furthermore, we compared the detection capabilities of specific HLA-B*15:01-restricted T cells using the HLA-B*15:01 and HLA-B*15:02 major histocompatibility complex (MHC) multimers and found remarkable differences in the staining patterns detected by flow cytometry. These new conditional ligands greatly add to the application of MHC-based technologies in the analyses of T-cell recognition as they represent frequently expressed HLA-B molecules. This expansion of conditional ligands is important to allow T-cell detection over a wide range of HLA restrictions, and provide comprehensive understanding of the T-cell recognition in a given context.

The interaction of KIR3DL1*001 with HLA class I molecules is dependent upon molecular microarchitecture within the Bw4 epitope.

The killer cell Ig-like receptor 3DL1 (KIR3DL1) inhibits activation of NK cells upon interaction with HLA class I molecules such as HLA-B*57:01, which contains the Bw4 epitope spanning residues 77-83 (e.g., NLRIALR), and not with HLA allomorphs that possess the Bw6 motif (e.g., HLA-B*08:01), which differ at residues 77, 80, 81, 82, and 83. Although Bw4 residues Ile(80) and Arg(83) directly interact with KIR3DL1*001, their precise role in determining KIR3DL1-HLA-Bw4 specificity remains unclear. Recognition of HLA-B*57:01 by either KIR3DL1(+) NK cells or the NK cell line YTS transfected with KIR3DL1*001 was impaired by mutation of residues 80 and 83 of HLA-B*57:01 to the corresponding amino acids within the Bw6 motif. Conversely, the simultaneous introduction of three Bw4 residues at positions 80, 82, and 83 into HLA-B*08:01 conferred an interaction with KIR3DL1*001. Structural analysis of HLA-B*57:01, HLA-B*08:01, and mutants of each bearing substitutions at positions 80 and 83 revealed that Ile(80) and Arg(83) within the Bw4 motif constrain the conformation of Glu(76), primarily through a salt bridge between Arg(83) and Glu(76). This salt bridge was absent in HLA-Bw6 molecules as well as position 83 mutants of HLA-B*57:01. Mutation of the Bw4 residue Ile(80) also disrupted this salt bridge, providing further insight into the role that position 80 plays in mediating KIR3DL1 recognition. Thus, the strict conformation of HLA-Bw4 allotypes, held in place by the Glu(76)-Arg(83) interaction, facilitates KIR3DL1 binding, whereas Bw6 allotypes present a platform on the α1 helix that is less permissive for KIR3DL1 binding.

Molecular imprint of exposure to naturally occurring genetic variants of human cytomegalovirus on the T cell repertoire.

Exposure to naturally occurring variants of herpesviruses in clinical settings can have a dramatic impact on anti-viral immunity. Here we have evaluated the molecular imprint of variant peptide-MHC complexes on the T-cell repertoire during human cytomegalovirus (CMV) infection and demonstrate that primary co-infection with genetic variants of CMV was coincident with development of strain-specific T-cell immunity followed by emergence of cross-reactive virus-specific T-cells. Cross-reactive CMV-specific T cells exhibited a highly conserved public T cell repertoire, while T cells directed towards specific genetic variants displayed oligoclonal repertoires, unique to each individual. T cell recognition foot-print and pMHC-I structural analyses revealed that the cross-reactive T cells accommodate alterations in the pMHC complex with a broader foot-print focussing on the core of the peptide epitope. These findings provide novel molecular insight into how infection with naturally occurring genetic variants of persistent human herpesviruses imprints on the evolution of the anti-viral T-cell repertoire.

A new HLA allele, HLA-B*08:108, described in two unrelated Spanish individuals.

HLA-B*08:108 shows one nucleotide difference regarding B*08:01:01 at codon 109 (CTC>TTC, L109>F109).

Mixed functional characteristics correlating with TCR-ligand koff -rate of MHC-tetramer reactive T cells within the naive T-cell repertoire.

The low frequency of antigen-specific naïve T cells has challenged numerous laboratories to develop various techniques to study the naïve T-cell repertoire. Here, we combine the generation of naïve repertoire-derived antigen-specific T-cell lines based on MHC-tetramer staining and magnetic-bead enrichment with in-depth functional assessment of the isolated T cells. Cytomegalovirus (CMV) specific T-cell lines were generated from seronegative individuals. Generated T-cell lines consisted of a variety of immunodominant CMV-epitope-specific oligoclonal T-cell populations restricted to various HLA-molecules (HLA-A1, A2, B7, B8, and B40), and the functional and structural avidity of the CMV-specific T cells was studied. Although all CMV-specific T cells were isolated based on their reactivity toward a specific peptide-MHC complex, we observed a large variation in the functional avidity of the MHC-tetramer positive T-cell populations, which correlated with the structural avidity measured by the recently developed Streptamer koff -rate assay. Our data demonstrate that MHC-tetramer staining is not always predictive for specific T-cell reactivity, and challenge the sole use of MHC-tetramers as an indication of the peripheral T-cell repertoire, independent of the analysis of functional activity or structural avidity parameters.

Autoimmune polyglandular syndromes: interplay between the immune and the endocrine systems leading to a diverse set of clinical diseases and new insights into immune regulation.

During the last 50 years, three major classes of autoimmune polyglandular syndromes (APSs) have been defined, and their characteristics and heritability have been delineated. Simultaneously, studies of the immunologic bases of these syndromes provided fundamental information in understanding immune regulation. Genetic analyses of patients and their families with APS type 1 (autoimmune polyendocrinopathy candidiasis, ectodermal dystrophy) identified the autoimmune regulator (AIRE) gene, which drives the expression of peripheral tissue-specific antigens in thymic cells and is critical in the development of self-tolerance. Mutations in this gene cause APS type 1. In contrast, studies in APS type 2 have been instrumental in understanding the role of human leukocyte antigen type II and related molecules in the pathogenesis of polygenetic autoimmune diseases such as type 1A diabetes. Immune dysfunction polyendocrinopathy, enteropathy, X-linked syndrome, which is caused by mutations in the forkhead box P3 gene, has been a model for studying regulatory T cell biology. The APSs epitomize the synergies that the merger of clinical and basic science can achieve. This is the environment that George Eisenbarth was able to create at the Barbara Davis Center for Diabetes.

Major histocompatibility complex class I chain related gene-A microsatellite polymorphism shows secondary association with type 1 diabetes and celiac disease in North Indians.

Microsatellite polymorphism in exon 5 of major histocompatibility complex class I chain related gene-A (MIC-A) has been implicated in the etiology of autoimmune diseases including type 1 diabetes (T1D) and celiac disease (CD). In this study on North Indian population, the MIC-A5.1 allele, carrying a premature termination codon in transmembrane region, was observed with increased frequency in T1D (29.6%, odds ratio OR = 2.1, P = 0.00017) and CD patients (40.3%, OR = 3.37, P = 1.67E-05) than in controls (16.7%). When the MIC-A5.1 association was adjusted for linkage with human leukocyte antigen (HLA)-DR3, the statistical significance of the association was abolished. This implies that the observed association of MIC-A5.1 is due to its linkage disequilibrium (D' = 0.94) with HLA-B8-DR3-DQ2 haplotype and is secondary to the overall association with DR3 positive MHC haplotypes.

HLA-B7-restricted EBV-specific CD8+ T cells are dysregulated in multiple sclerosis.

It was hypothesized that the EBV-specific CD8(+) T cell response may be dysregulated in multiple sclerosis (MS) patients, possibly leading to a suboptimal control of this virus. To examine the CD8(+) T cell response in greater detail, we analyzed the HLA-A2-, HLA-B7-, and HLA-B8-restricted EBV- and CMV-specific CD8(+) T cell responses in a high number of MS patients and control subjects using tetramers. Content in cytolytic granules, as well as cytotoxic activity, of EBV- and CMV-specific CD8(+) T cells was assessed. We found that MS patients had a lower or a higher prevalence of HLA-A2 and HLA-B7, respectively. Using HLA class I tetramers in HLA-B7(+) MS patients, there was a higher prevalence of MS patients with HLA-B*0702/EBV(RPP)-specific CD8(+) T cells ex vivo. However, the magnitude of the HLA-B*0702/EBV(RPP)-specific and HLA-B*0702/CMV(TPR)-specific CD8(+) T cell response (i.e., the percentage of tetramer(+) CD8(+) T cells in a study subject harboring CD8(+) T cells specific for the given epitope) was lower in MS patients. No differences were found using other tetramers. After stimulation with the HLA-B*0702/EBV(RPP) peptide, the production of IL-2, perforin, and granzyme B and the cytotoxicity of HLA-B*0702/EBV(RPP)-specific CD8(+) T cells were decreased. Altogether, our findings suggest that the HLA-B*0702-restricted viral (in particular the EBV one)-specific CD8(+) T cell response is dysregulated in MS patients. This observation is particularly interesting knowing that the HLA-B7 allele is more frequently expressed in MS patients and considering that EBV is associated with MS.

A 2 amino acid shift in position leads to a substantial difference in the pattern of processing of 2 HIV-1 epitopes.

BACKGROUND: The sequence diversity that exists between HIV-1 strains presents a major obstacle to the design of a vaccine that will be effective on a global scale. Focusing on highly conserved cytotoxic T-lymphocyte epitopes as vaccine targets has been called into question by evidence that variation within epitope flanking regions can affect processing and presentation. METHODS: Using epitope-specific T-cell clones tested for recognition of HLA-matched target cells infected with vaccinia viruses expressing HIV-1 nef genes derived from different HIV-1 clades, we examined the efficiency of presentation of an HLA-B*40 restricted HIV-1 nef epitope compared to that of an HLA-B*08 restricted epitope with which it overlaps by 6 amino acides. RESULTS: This small shift in epitope position substantially changed the patter or epitope processing and led either to an increase or decrease in antigen generation dependent on the viral sequences present. CONCLUSIONS: These data demonstrate the complexity of the antigen presentation pathway and the difficulties associated with selecting CTL epitopes as targets for an HIV-1 vaccine that will be effective in many populations and against several viral strains.

A structural basis for varied αß TCR usage against an immunodominant EBV antigen restricted to a HLA-B8 molecule.

EBV is a ubiquitous and persistent human pathogen, kept in check by the cytotoxic T cell response. In this study, we investigated how three TCRs, which differ in their T cell immunodominance hierarchies and gene usage, interact with the same EBV determinant (FLRGRAYGL), bound to the same Ag-presenting molecule, HLA-B8. We found that the three TCRs exhibit differing fine specificities for the viral Ag. Further, via structural and biophysical approaches, we demonstrated that the viral Ag provides the greatest energetic contribution to the TCR-peptide-HLA interaction, while focusing on a few adjacent HLA-based interactions to further tune fine-specificity requirements. Thus, the TCR engages the peptide-HLA with the viral Ag as the main glue, such that neighboring TCR-MHC interactions are recruited as a supportive adhesive. Collectively, we provide a portrait of how the host's adaptive immune response differentially engages a common viral Ag.

Quantitative and functional diversity of cross-reactive EBV-specific CD8+ T cells in a longitudinal study cohort of lung transplant recipients.

BACKGROUND: Cross-reactive antiviral memory T cells constitute a significant proportion of the alloresponse, potentially playing a pivotal role in adverse posttransplant outcomes in human leukocyte antigen (HLA)-mismatched allografts. We explored the longitudinal dynamics of cross-reactive HLA-B8-restricted Epstein-Barr virus-specific CD8+ T cells directed toward the EBNA3A epitope FLRGRAYGL (FLR) in lung transplant recipients (LTRs) to determine whether their corecognition of HLA-B*4402 expressed on the allograft contributed to poorer posttransplant outcomes. METHODS: Cross-reactive FLR-specific CD8+ T cells were measured in the peripheral blood mononuclear cells and bronchoalveolar lavage fluid in 11 HLA-B8+ LTR, who had received HLA-B44+ lung allograft, after in vitro autologous (FLR pulsed) or allogeneic stimulation by multiparameter flow cytometry. RESULTS: FLR-specific CD8+ T cells were detectable ex vivo and after 13 days following in vitro peptide stimulation of peripheral blood mononuclear cells. Individual LTR and demonstrated diverse functional profiles of either cytokine production and/or cytotoxic potential (interferon-g+, interferon-g+CD107a+ and CD107a+ subsets). However, cells isolated from bronchoalveolar lavage exhibited a skewed functional phenotype toward CD107a expression alone, indicating cytotoxic-producing but not cytokine-producing capabilities. In addition, our findings suggested that the presence of cross-reactive FLR-specific CD8+ T cells may influence the alloreactive hierarchy directed against the allograft, although they were not associated with poorer short- or long-term clinical outcomes in the absence of Epstein-Barr virus reactivation and in the setting of current immunosuppression and antiviral prophylaxis protocols. CONCLUSION: We report, for the first time, the longitudinal measurement of cross-reactive FLR-specific CD8 T cells within a clinical transplantation framework.