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.

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.

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).

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 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.

HLA-B8, DR3: a new risk factor for graft failure after renal transplantation in patients with underlying immunoglobulin A nephropathy.

BACKGROUND: The HLA-B8, DR3 haplotype has been associated with high immune reactivity. In this study, we have tested whether this haplotype has differential effect on graft survival in patients with IgAN compared with control patients. METHODS: From the Eurotransplant Registry we analyzed graft survival of 1207 recipients with IgAN and 7935 control patients with non-glomerular diseases. Death-censored graft loss according to the HLA-B8, DR3 haplotype was calculated with Kaplan-Meier analysis and Cox-regression model was used to correct for various risk factors. RESULTS: The frequency of the HLA-B8, DR3 haplotype was significantly lower in IgAN patients compared with controls (10.3% vs. 15.4%, p < 0.001). Ten-year graft survival was identical in the control group with and without the HLA-B8, DR3 haplotype (71.1% and 70.2%, respectively), but significantly worse in IgAN patients carrying the HLA-B8, DR3 haplotype compared with patients without it (52.5% vs. 69.1%, respectively, p = 0.009). The risk of graft loss was increased by 66% (HR 1.6, 95% CI 1.14, 2.29) in IgAN with the HLA-B8, DR3 haplotype and independent of well-known risk factors. CONCLUSIONS: We have identified a new risk factor for graft loss unique to patients with IgAN. This finding emphasizes the exclusive immune characteristics of IgAN patients after transplantation.

The shaping of T cell receptor recognition by self-tolerance.

During selection of the T cell repertoire, the immune system navigates the subtle distinction between self-restriction and self-tolerance, yet how this is achieved is unclear. Here we describe how self-tolerance toward a trans-HLA (human leukocyte antigen) allotype shapes T cell receptor (TCR) recognition of an Epstein-Barr virus (EBV) determinant (FLRGRAYGL). The recognition of HLA-B8-FLRGRAYGL by two archetypal TCRs was compared. One was a publicly selected TCR, LC13, that is alloreactive with HLA-B44; the other, CF34, lacks HLA-B44 reactivity because it arises when HLA-B44 is coinherited in trans with HLA-B8. Whereas the alloreactive LC13 TCR docked at the C terminus of HLA-B8-FLRGRAYGL, the CF34 TCR docked at the N terminus of HLA-B8-FLRGRAYGL, which coincided with a polymorphic region between HLA-B8 and HLA-B44. The markedly contrasting footprints of the LC13 and CF34 TCRs provided a portrait of how self-tolerance shapes the specificity of TCRs selected into the immune repertoire.

Genetic association of autoimmune hepatitis and human leucocyte antigen in German patients.

AIM: To report on our large German collective and updated data of 142 patients with autoimmune hepatitis (AIH) type 1. METHODS: Key investigations performed were liver biopsy, serum autoantibodies as well as serum markers such as IgG and elevated transaminases. Antinuclear antigen (ANA) and smooth muscle antigen (SMA) autoantibodies characterized type 1 AIH. Type 3 (AIH) was solely characterized by the occurrence of soluble liver antigen/liver-pancreas antigen (SLA/LP) autoantibodies either with or without ANA or SMA autoantibodies. RESULTS: Most prevalent HLAs were A2 (68 patients, 48%), B8 (63 patients, 44%), C7 (90 patients, 63%), DR3 (49 patients, 38%), DR4 (49 patients, 38%) and DQ2 (42 patients, 30%). Compared to the Italian and North American patients, we found fewer patients with a DQ2 subtype. Furthermore, the B8-DR3-DQ2 human leucocyte antigen (HLA) was also less prominent compared to the North American patients. However, prevalences of B8, DR3, DR4, DR7, DR11 and DR13 were comparable to the Italian and North American patients. Furthermore, we report on an additional subgroup of patients with SLA/LP positive AIH. Generally, in this subgroup of patients the same HLA subtypes were favoured as the AIH type 1. CONCLUSION: Although HLA subtypes were comparable between these three collectives, the German patients were distinct from the Italian and North American patients with respect to DQ2 and from the North American patients with respect to B8-DR3-DQ2 HLA. A clinical correlation, e.g. difference in severity or treatability of AIH type 1, has yet to be determined.

HLA-B8 association with late-stage melanoma--an immunological lesson?

BACKGROUND: Differences in HLA allele frequencies between the diseased and healthy populations may signify efficient immune responses, a notion that has been successfully tested for infectious diseases or for association with genetic elements involved in a distinct type of immunity. This retrospective study is intended to detect differences in MHC class I carrier frequencies of advanced melanoma patients compared to healthy bone marrow donors. METHODS: The HLA-A and -B carrier frequencies of 748 stage IV melanoma patients retrieved from serotyping at 6 different centers in Germany were compared using a chi-square test to 13,386 fully HLA typed bone marrow donors registered in the German national bone marrow donor registry. RESULTS: The comparison of HLA carrier frequencies in advanced cancer patients with healthy bone marrow donors revealed a significant decrease in HLA-B8 carrier frequencies, which was also apparent in patients with advanced disease compared to patients with loco-regional disease. CONCLUSION: The data suggest that protective immune responses restricted to distinct MHC class I molecules may be operational in a subset of melanoma patients, which is the prerequisite for a large scale screen for the corresponding epitopes. Alternatively, the known association of the ancestral haplotype HLA-A1, -B8 and -DR3 with genetic elements such as distinct TNF-alpha alleles might have a protective effect on disease progression. In any case, identification of the cause of protection within this patient subset might lead to a significant improvement in the efficacy of current immunotherapeutic approaches.

T cell epitope definition by differential mass spectrometry: identification of a novel, immunogenic HLA-B8 ligand directly from renal cancer tissue.

In this study, we describe a differential mass spectrometric technique for the immuno-proteomic analysis of the major histocompatibility complex (MHC) peptides of a renal cell carcinoma (RCC) biopsy compared with the healthy kidney tissue of the same patient after nephrectomy. Using a stable isotope labeling approach, we could directly compare and relatively quantify 43 MHC-peptide pairs, most of which were present in similar proportions on both normal kidney and tumor. Significantly, two dominant peptides of monoisotopic masses ([M+H](+)) 973.43 u and 967.59 u, respectively, were found exclusively in the tumor sample. One of these was identified as originating from heme oxygenase-1 (HO-1), a protein involved in induction of apoptosis resistance, immuno-suppression and neoangiogenesis and reported to be up-regulated in various cancer types. Moreover, the corresponding synthetic HO-1-derived peptide was shown to be immunogenic in vitro by generation of CD8+ T cell lines with peptide-specific cytolytic activity. Thus, this peptide is an example of a differentially identified T cell epitope that could be considered as a target for immunotherapy.

Antagonism of antiviral and allogeneic activity of a human public CTL clonotype by a single altered peptide ligand: implications for allograft rejection.

Alloreactive T lymphocytes are central mediators of graft-versus-host disease and allograft rejection. A public CTL clonotype with specificity for the alloantigens HLA-B*4402 and B*4405 is often expanded to large numbers in healthy HLA-B*0801(+) individuals, driven by cross-reactive stimulation with the common, persistent herpesvirus EBV. Since such alloreactive memory CTL expansions have the potential to influence transplantation outcome, altered peptide ligands (APLs) of the target HLA-B*0801-binding EBV peptide, FLRGRAYGL, were screened as specific antagonists for this immunodominant clonotype. One APL, FLRGRFYGL, exerted powerful antagonism of a prototypic T cell clone expressing this immunodominant TCR when costimulated with target cells presenting HLA-B*0801(FLRGRAYGL). Significantly, this APL also reduced the lysis of allogeneic target cells expressing HLA-B*4402 by up to 99%. The affinities of the agonist and antagonist complexes for the public TCR, measured using solution and solid-phase assays, were 8 and 138 muM, respectively. Surprisingly, the half-life of the agonist and antagonist complexes was similar, yet the association rate for the antagonist complex was significantly slower. These observations were further supported by structural studies that suggested a large conformational hurdle was required to ligate the immunodominant TCR to the HLA-B*0801 antagonist complex. By defining an antagonist APL against an immunodominant alloreactive TCR, these findings raise the prospect of exploiting such peptides to inhibit clinical alloreactivity, particularly against clonal T cell expansions that react with alloantigens.

A Gambian TNF haplotype matches the European HLA-A1,B8,DR3 and Chinese HLA-A33,B58,DR3 haplotypes.

Caucasians carry TNFA-308*2 in the 8.1 ancestral haplotype (AH) (HLA-A1,B8,DR3). In Gambians, TNFA-308*2 occurs without HLA-B8 or -DR3, suggesting an independent effect of TNFA-308 on disease. Hence we sought a segment of the 8.1 AH in Gambians. BAT1 (intron 10)*2 was selected as a specific marker of the haplotype and was found with TNFA-308*2 in Gambians. Samples homozygous at TNFA-308 and BAT1 (intron 10) demonstrated identity between the African TNFA-308*2 haplotype, the 8.1AH and the Asian diabetogenic 58.1AH (HLA-A33,B58,DR3) across a region spanning BAT1, ATP6G, IKBL, LTA, TNFA, LTB, LST-1 and AIF-1. Conservation of this block in geographically distinct populations suggests a common evolutionary origin and challenges current views of the role of TNFA-308*2 in disease.

Alleles of the proximal promoter of BAT1, a putative anti-inflammatory gene adjacent to the TNF cluster, reduce transcription on a disease-associated MHC haplotype.

BACKGROUND: BAT1 belongs to the DEAD-box family of proteins, and is encoded in the central region of the MHC, a region containing genes affecting immunopathological disorders including Type 1 diabetes. We showed that BAT1 can reduce inflammatory cytokine production, supporting its candidacy as a disease gene. Here we examined the proximal promoter region of BAT1. RESULTS: Ten single nucleotide polymorphisms were identified in approximately 1.4 kb of sequence, defining at least seven alleles. Sections of the BAT1 promoter region were amplified from cells homozygous for the MHC haplotypes associated with susceptibility (HLA-A1, B8, DR3; 8.1 haplotype) and resistance (HLA-A3, B7, DR15; 7.1 haplotype) to diabetes and cloned into a promoter-less luciferase-encoding plasmid. Jurkat cells transiently transfected with fragments from the 8.1 haplotype exhibited a lower luciferase activity than those transfected with fragments from the 7.1 haplotype, indicating reduced transcription. The effect was clearest with the 520 bp immediately upstream of the transcriptional start site. Electrophoretic mobility shift assays using oligonucleotides spanning polymorphic sites within the 520 bp (proximal) promoter fragment showed haplotype-specific binding of nuclear proteins. CONCLUSIONS: In view of the anti-inflammatory role of BAT1, reduced production on a disease-associated haplotype constitutes a novel and self-consistent model for the effect of central MHC genes on disease.