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Adoptively transferred tumor-infiltrating T lymphocytes (TILs) that mediate complete regression of metastatic melanoma have been shown to recognize mutated epitopes expressed by autologous tumors. Here, in an attempt to develop a strategy for facilitating the isolation, expansion, and study of mutated antigen–specific T cells, we performed whole-exome sequencing on matched tumor and normal DNA isolated from 8 patients with metastatic melanoma. Candidate mutated epitopes were identified using a peptide-MHC–binding algorithm, and these epitopes were synthesized and used to generate panels of MHC tetramers that were evaluated for binding to tumor digests and cultured TILs used for the treatment of patients. This strategy resulted in the identification of 9 mutated epitopes from 5 of the 8 patients tested. Cells reactive with 8 of the 9 epitopes could be isolated from autologous peripheral blood, where they were detected at frequencies that were estimated to range between 0.4% and 0.002%. To the best of our knowledge, this represents the first demonstration of the successful isolation of mutation-reactive T cells from patients’ peripheral blood prior to immune therapy, potentially providing the basis for designing personalized immunotherapies to treat patients with advanced cancer.

Immune therapy has provided a significant breakthrough in the treatment of metastatic melanoma. Despite the remarkable clinical efficacy and established involvement of effector CD8 T cells, the knowledge of the exact peptide-MHC complexes recognized by T cells on the tumor cell surface is limited. Many melanoma-associated T-cell epitopes have been described, but this knowledge remains largely restricted to HLA-A2, and we lack understanding of the T-cell recognition in the context of other HLA molecules. We selected six melanoma-associated antigens (MAGE-A3, NY-ESO-1, gp100, Mart1, tyrosinase and TRP-2) that are frequently recognized in patients with the aim of identifying novel T-cell epitopes restricted to HLA-A1, -A3, -A11 and -B7. Using in silico prediction and in vitro confirmation, we identified 127 MHC ligands and analyzed the T-cell responses against these ligands via the MHC multimer-based enrichment of peripheral blood from 39 melanoma patients and 10 healthy donors. To dissect the T-cell reactivity against this large peptide library, we used combinatorial-encoded MHC multimers and observed the T-cell responses against 17 different peptide-MHC complexes in the patient group and four in the healthy donor group. We confirmed the processing and presentation of HLA-A3-restricted T-cell epitopes from tyrosinase (TQYESGSMDK) and gp100 (LIYRRRLMK) and an HLA-A11-restricted T-cell epitope from gp100 (AVGATKVPR) via the cytolytic T-cell recognition of melanoma cell lines and/or K562 cells expressing the appropriate antigen and HLA molecule. We further found T-cell reactivity against two of the identified sequences among tumor-infiltrating lymphocytes from melanoma patients, suggesting a potential clinical relevance of these sequences. Electronic supplementary material The online version of this article (doi:10.1007/s00262-015-1664-x) contains supplementary material, which is available to authorized users.

Peptides are extensively used to characterize functional or (linear) structural aspects of receptor–ligand interactions in biological systems, e.g. SH2, SH3, PDZ peptide-recognition domains, the MHC membrane receptors and enzymes such as kinases and phosphatases. NNAlign is a method for the identification of such linear motifs in biological sequences. The algorithm aligns the amino acid or nucleotide sequences provided as training set, and generates a model of the sequence motif detected in the data. The webserver allows setting up cross-validation experiments to estimate the performance of the model, as well as evaluations on independent data. Many features of the training sequences can be encoded as input, and the network architecture is highly customizable. The results returned by the server include a graphical representation of the motif identified by the method, performance values and a downloadable model that can be applied to scan protein sequences for occurrence of the motif. While its performance for the characterization of peptide–MHC interactions is widely documented, we extended NNAlign to be applicable to other receptor–ligand systems as well. Version 2.0 supports alignments with insertions and deletions, encoding of receptor pseudo-sequences, and custom alphabets for the training sequences. The server is available at http://www.cbs.dtu.dk/services/NNAlign-2.0. Fil: Nielsen, Morten. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina. Technical University of Denmark; Dinamarca Fil: Andreatta, Massimo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina

Objective: Familial clustering of juvenile autoimmune liver disease (AILD), including autoimmune hepatitis and autoimmune sclerosing cholangitis (ASC), is rare, despite a high prevalence of autoimmune disorders in AILD families. Methods: To investigate this discrepancy, we measured autoantibodies diagnostic for AILD, anti-nuclear, anti-smooth muscle, anti-liver kidney microsomal type 1, anti-liver cytosol type 1, and anti-soluble liver antigen antibodies, and human leukocyte antigen profiles in 31 patients and 65 of their first-degree relatives (FDR). The autoantibody profile was compared with that of 42 healthy subjects (HS). Results: Autoantibodies were detected in 71% (22/31) patients. Antinuclear antibody or anti-smooth muscle antibody were present in 4/65 FDR (6.2%). HS were negative for all autoantibodies. The frequencies of homozygous HLA DRB1� 0301 (DR3) genes and haplotype A1-B8-DR3 were higher in the patients (25% and 43%) than in FDR (9% and 27%) and HS (0% and 16%). The frequencies of disease-protective genes DR4 and/or DR15 were lower in the patients (25%) than in FDR (42%) and HS (42%). Only 1 family contained 2 patients with AILD, 1 with ASC and 1 with primary sclerosing cholangitis. Both patients possessed A1-B8-DR3 genes, the ASC being homozygous and the primary sclerosing cholangitis heterozygous. Six FDR had nonhepatic autoimmune disorders, none being autoantibody positive. Conclusions: Homozygosity for DR3 plays a major role in the predisposition to juvenile AILD. Diagnostic autoantibodies for AILD are rare among patients’ FDR and not linked to clinical manifestation of AILD.

Malignant melanoma, a very common type of cancer, is a rapidly growing cancer of the skin with an increase in incidence among the Caucasian population. The disease is seen through all age groups and is very common in the younger age groups. Several studies have examined the risk factors and pathophysiological mechanisms of malignant melanoma, which have enlightened our understanding of the development of the disease, but we have still to fully understand the complex immunological interactions. The examination of the interaction between the human leucocyte antigen (HLA) system and prognostic outcome has shown interesting results, and a correlation between the down- or upregulation of these antigens and prognosis has been seen through many different types of cancer. In malignant melanoma, HLA class Ia has been seen to influence the effects of pharmaceutical drug treatment as well as the overall prognosis, and the HLA class Ib and regulatory T cells have been correlated with tumor progression. Although there is still no standardized immunological treatment worldwide, the interaction between the human leucocyte antigen (HLA) system and tumor progression seems to be a promising focus in the way of optimizing the treatment of malignant melanoma.

HLA-A*01:234 was identified by next-generation sequencing and confirmed by Sanger sequencing.

One nucleotide changes in position 740 of HLA-A*01:01 result in a novel null-allele, HLA-A*01:308N.

We describe an HLA-A1 melanoma patient who has mounted a spontaneous cytolytic T cell (CTL) response against an antigenic peptide encoded by gene MAGE-A3 and presented by HLA-A1. The frequency of anti-MAGE-3.A1 CTLp was 5 x 10(-7) of the blood CD8 cells, with a dominant clonotype which was present in six out of seven independent anti-MAGE-3.A1 CTL clones. After vaccination with a recombinant poxvirus coding for the MAGE-3.A1 antigen, the blood frequency of anti-MAGE-3.A1 CTLp increased tenfold. Twenty-two independent CTL clones were derived. Surprisingly, only one of them corresponded to the dominant clonotype present before vaccination. Two new clonotypes were repeated 12 and 7 times, respectively. Our interpretation of these results is that the spontaneous anti-MAGE-3.A1 CTL response pre-existing to vaccination was polyclonal, and that the vaccine restimulated only some of these clones. To estimate the incidence of spontaneous anti-MAGE-3.A1 CTL responses in melanoma patients with a tumor expressing gene MAGE-A3, we measured the blood frequency of anti-MAGE-3.A1 T cells in 45 patients, and found only two clear responses.

Immunogenetic information is provided on five novel alleles with synonymous mutations.

Failure of the adaptive immune response to control infection with the hepatitis C virus (HCV) can result from mutational escape in targeted T-cell epitopes. Recent studies suggest that T-cell immune pressure is an important factor in the evolution of the nonstructural proteins in HCV. The aim of this study was to characterize the forces that contribute to viral evolution in an HLA-A*01-restricted epitope in HCV NS3. This epitope represents a potentially attractive target for vaccination strategies since it is conserved across all genotypes. In our cohort of subjects with chronic HCV infection (genotype 1b or 3a), it is a frequently recognized CD8 epitope in HLA-A*01-positive subjects. Viral sequence data reveal that an escape variant is the dominant residue in both genotypes. The predominant Y1444F substitution seemingly impairs binding to the HLA-A*01 molecule, which may have an important impact on the ability to prime a functional CD8 response upon infection. Interestingly, a case of evolution toward the prototype sequence was observed during chronic infection, possibly because the helicase activity of the protein containing the Y1444F substitution is reduced compared to the prototype sequence. Comparison of HCV sequences from Asia and Europe suggests that the frequency of the HLA-A*01 allele in a population may influence the frequency of the escape variant in circulating strains. These data suggest a complex interaction of multiple forces shaping the evolution of HCV in which immune pressure both within the individual and also at the population level in addition to functional constraints are important contributing factors.

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 (

This study was a retrospective analysis of Thai patients undergoing T-replete hematopoietic stem cell transplant from human leukocyte antigen (HLA)-identical sibling donors. We investigated 66 patients, including 40 patients with acute myeloid leukemia (AML), 12 patients with acute lymphoblastic leukemia and 14 patients with chronic myeloid leukemia. Killer cell immunoglobulin-like receptor (KIR) genes and HLA ligands were typed by polymerase chain reaction-sequence specific oligonucleotide probes. We analyzed the effect of the number of missing KIR ligands (Bw4, C1 and C2) on clinical outcomes. A beneficial effect of missing KIR ligand was not observed in both univariate and multivariate analysis. When we analyzed the effect of specific missing KIR ligand on clinical outcomes, there was a trend that patients with missing A11 ligand had lower relapse rate (P = 0.076). Therefore, we also conducted the analysis by including the group with missing KIR ligands of Bw4, C1, C2 and A11. Patients with two or more than two missing KIR ligands had a trend for better clinical outcome including reduced relapse (P = 055) and statistically significant in terms of reduced acute graft-vs-host disease (aGVHD) rate (P = 0.013). In multivariate analysis, patients with two or more than two missing KIR ligands had a statistically significant better clinical outcome in terms of reduced aGVHD rate (HR = 0.155, 95%CI = 0.040-0.605, P = 0.007). The association between clinical outcome with KIR haplotypes, centromeric B haplotype and activating KIR was not observed here. Although the sample size in this study is rather limited, these data can later be subjected to meta-analysis to help reach the conclusion of the usefulness of this additional promising KIR genotyping in various hematopoietic stem cell transplantation types.

Severe forms of dengue, such as dengue haemorrhagic fever (DHF) and dengue shock syndrome, are examples of a complex pathogenic mechanism in which the virus, environment and host immune response interact. The influence of the host's genetic predisposition to susceptibility or resistance to infectious diseases has been evidenced in several studies. The association of the human leukocyte antigen gene (HLA) class I alleles with DHF susceptibility or resistance has been reported in ethnically and geographically distinct populations. Due to these ethnic and viral strain differences, associations occur in each population, independently with a specific allele, which most likely explains the associations of several alleles with DHF. As the potential role of HLA alleles in the progression of DHF in Brazilian patients remains unknown, we then identified HLA-A alleles in 67 patients with dengue fever and 42 with DHF from Rio de Janeiro, Brazil, selected from 2002-2008 by the sequence-based typing technique. Statistical analysis revealed an association between the HLA-A*01 allele and DHF [odds ratio (OR) = 2.7, p = 0.01], while analysis of the HLA-A*31 allele (OR = 0.5, p = 0.11) suggested a potential protective role in DHF that should be further investigated. This study provides evidence that HLA class I alleles might be important risk factors for DHF in Brazilian patients.

We present human leukocyte antigen (HLA) haplotype and allele/antigenic group frequencies derived from a data set of 920 umbilical cord blood units collected in Central Chile. HLA-A and -B genotypes were typed using sequence specific oligonucleotide probe methods while HLA-DRB1 genotypes were obtained from sequencing-based typing. The most frequent haplotype is A*29~B*44~DRB1*07:01 with an estimated frequency of 2.1%.

Accurately predicting peptides binding to major histocompatibility complex (MHC) I molecules is of great importance to immunologists for elucidating the underlying mechanism of immune recognition and facilitating the design of peptide-based vaccine. Various computational methods have been developed for MHC I-peptide binding prediction, and several of them are reported to achieve high accuracy in recent evaluation on benchmark datasets. For attending the machine learning in immunology competition (MLIC) in prediction of human leukocyte antigen (HLA)-binding peptides, we (FudanCS) have made use of ensemble approaches to further improve the prediction performance by integrating the outputs of several leading predictors. Two ensemble approaches, PM and AvgTanh, have been implemented for attending MLIC. AvgTanh and PM achieved the fourth and the seventh out of all 20 submissions in MLIC in terms of the average AUC. In addition, AvgTanh was awarded the winner in the category of HLA-A*0101 of 9-mer. Overall, the competition results validate the effectiveness of ensemble approaches.

There has been considerable interest in statistical approaches that leverage the large volumes of experimental data to predict the binding of Major Histocompatibility Complex class I (MHC-I) molecules to peptides. Here we present our method for averaging together multiple predictors for MHC-peptide binding, where given a particular MHC molecule, a set of predictors and a set of training peptides, our method will average multiple simple predictors for MHC binding to produce a final prediction of the binding affinity between a given MHC molecule and a test peptide. The averaging of predictors is done using a nonparametric method, whereby for any test peptide, we identify similar peptides in the training set and average the predictions on the training set, weighted by each predictor's average accuracy for similar peptides in the training set. We show that our method significantly improves on individual predictors based on held-out data and also produces a predictor whose accuracy is competitive with state-of-the-art techniques based on the results from the Machine Learning in Immunology competition in which 21 submitted techniques were assessed on their accuracy in predicting the binding of HLA-A*0101, HLA-A*0201 and HLA-B*0702 molecules to 9-mer and 10-mer peptides.

Cytomegalovirus (CMV) infection is an important cause of morbidity and mortality in hematopoietic stem cell transplant recipients despite the introduction of posttransplantation viral monitoring and preemptive antiviral therapy. We evaluated the use of HLA class I tetramers in monitoring CMV-specific T-cell recovery to predict patients at risk for CMV-related complications. This prospective multicenter clinical trial obtained nearly 1400 tetramer/allele results in more than 800 biweekly blood samples from 83 patients monitored for 1 year after transplantation. Major HLA types were included (A*0101, A*0201, B*0702, B*0801, B*3501). iTAg MHC Tetramers (Beckman Coulter) were used to enumerate CMV-specific CD8+ T cells by flow cytometry using a single-platform absolute counting method. Assay variability was 8% or less and results were available within 3 hours. Delayed recovery of CMV-specific T cells (< 7 cells/μL in all blood samples during the first 65 days after transplantation) was found to be a significant risk factor for CMV-related complications; these patients were more likely to develop recurrent or persistent CMV infection (relative risk 2.6, CI 1.2-5.8, P = .01) than patients showing rapid recovery, which was associated with protection from CMV-related complications (P = .004). CMV tetramer–based immune monitoring, in conjunction with virologic monitoring, can be an important new tool to assess risk of CMV-related complications and to guide preemptive therapeutic choices.

Immunotherapy of colorectal carcinoma (CRC) has great promise as the presence of T lymphocytes in CRC tissues in situ is correlated with reduced recurrence and increased survival. Thus, identification of the antigens recognized by T cells of CRC patients may permit development of vaccines with potential benefit for these patients. Using expression cloning, we identified the antigen, nucleophosmin (Npm), recognized by an HLA-A1 restricted cytotoxic T lymphocyte (CTL) line derived from the peripheral blood mononuclear cells (PBMC) of a rectal cancer patient. A decamer peptide derived from the Npm sequence sensitized peptide-pulsed HLA-A1 positive cells to lysis by the CTL line. The peptide also induced proliferative and cytotoxic T lymphocytes in the PBMC of 4 of 6 CRC patients, which lysed HLA-A1 positive peptide-pulsed target cells and CRC cells endogenously expressing Npm. Overexpression of Npm by tumors of various histological types, recognition of the antigen by T cells derived from different CRC patients and association of the antigen with poor prognostic outcome make it a promising target for immunotherapeutic intervention in cancer patients.

Background: Some cancer patients are immuno-compromised, and it has been long felt that immune-intervention is not compatible with standard chemotherapies. However, increasing evidence suggests that standard chemotherapy drugs may stimulate beneficial changes in both the immune system and tumour. Methods: We have assessed the expression of human leucocyte antigen class 1 (HLA1) on tumour cells before and after chemotherapy agents (cyclophosphamide, oxaliplatin or gemcitabine). In addition, we show that chemotherapy-stressed tumour cells may release cytokines that enhance the interactions between dendritic cells (DCs) and T cells into growth media. Results: Here we report that some chemotherapy agents can increase HLA1 expression in tumour cells, even when expression is low. Increases were associated with killing by cytotoxic T cells, which were negated by HLA1-blockade. Furthermore, T-cell function, as indicated by increased proliferation, was enhanced as supernatants derived from tumours treated with chemotherapy augmented DC-maturation and function. Conclusion: There is evidence that a facet of immune surveillance can be restored by appropriate chemotherapy agents. Also, tumours exposed to some chemotherapy may secrete cytokines that can mature DCs, which ultimately enhances T-cell responses.

Objectives. As the HLA system is involved in recognition of self and non-self, an association with the development of ANCA-associated vasculitis (AAV) seems probable. In this study, the relation between HLA antigens and AAV and its severity were investigated.Methods. Consecutive patients diagnosed with AAV at our centre, who were followed for at least 2 years, were included. The frequency of HLA antigens of AAV and WG patients was compared with 5872 healthy blood donors from the same region and with 4000 healthy Dutch controls originating from a Eurotransplant database.Results. From 304 AAV patients, sufficient data were available. We found DR13(6) to be less prevalent and both DR4 and the ancestral haplotype A1B8DR3 more prevalent in patients with AAV compared with controls, particularly in patients with WG. In addition, DR1 was less prevalent in patients with WG in comparison with controls. Further, DR8 was more prevalent in patients with CSS compared with other forms of vasculitis and controls. There were no associations between HLA antigens and disease characteristics or course of AAV or WG.Conclusions. AAV is associated with increased prevalence of DR4 and the ancestral haplotype A1B8DR3 and with decreased prevalence of DR13(6), particularly in patients with WG. In patients with WG, prevalence of DR1 was decreased, whereas in patients with CSS DR8 was increased. No associations between HLA antigens and disease characteristics or course of AAV were found.

Background: Interleukin-12 (IL-12) has been shown to enhance the cytotoxic activity of NK cells and CTL. IL-12 also acts as a growth factor for activated NK, T and NKT cells. The soluble HLA class I (sHLA-I) has been reported to bind a killer-cell inhibitory receptor, which is expressed on the NK cell, and its signals inhibit NK cell-mediated cytotoxicity. Effects of fresh frozen plasma (FFP) on post-operative immune status have not yet been completely examined. Methods: Thirty consecutive patients taking a hepatectomy were enrolled. The levels of IL-12 and sHLA-I were examined by enzyme-linked immunosorbent assay. Results: The rate of complication after hepatectomy in the FFP-administered patients was higher than that in patients without FFP administration (P= 0.0358). Decreased IL-12 levels after surgery in patients without FFP administration recovered to the preoperative state earlier than those in patients with FFP administration (P < 0.05). The levels of sHLA-I in the FFP-administered patients were higher than those in the patients without FFP administration (P < 0.05). Conclusions: Administration of FFP, which contains sHLA-I, affected the levels of sHLA-I after hepatectomy. Both high levels of sHLA-I and low levels of IL-12 could attenuate NK activities after hepatectomy, especially when FFP would be administered.

To present virus and tumor Ags, HLA class I molecules undergo a complex multistep assembly involving discrete but transient folding intermediates. The most extensive folding abnormalities occur in cells lacking the class I L chain subunit, called β2-microglobulin (β2m). Herein, this issue was investigated taking advantage of eight conformational murine mAbs (including the prototypic W6/32 mAb) to mapped H chain epitopes of class I molecules, four human mAbs to class I alloantigens, as well as radioimmunoprecipitation, in vitro assembly, pulse-chase, flow cytometry, and peptide-pulse/ELISPOT experiments. We show that endogenous (HLA-A1, -A66, and -B58) as well as transfected (HLA-A2) heavy chains in β2m-defective Burkitt lymphoma Daudi cells are capable of being expressed on the cell surface, although at low levels, and exclusively as immature glycoforms. In addition, HLA-A2 is: 1) partially folded at crucial interfaces with β2m, peptide Ag, and CD8; 2) receptive to exogenous peptide; and 3) capable of presenting exogenous peptide epitopes (from virus and tumor Ags) to cytotoxic T lymphocytes (bulk populations as well as clones) educated in a β2m-positive environment. These experiments demonstrate a precursor-product relationship between novel HLA class I folding intermediates, and define a stepwise mechanism whereby distinct interfaces of the class I H chain undergo successive, ligand-induced folding adjustments in vitro as well as in vivo. Due to this unprecedented class I plasticity, Daudi is the first human cell line in which folding and function of class I HLA molecules are observed in the absence of β2m. These findings bear potential implications for tumor immunotherapy.

S100A4 (metastasin 1) belongs to the S100 family of Ca(2+) binding proteins. While not present in most differentiated adult tissues, S100A4 is upregulated in the micromilieu of tumors. It is primarily expressed by tumor-associated macrophages, fibroblasts, and tumor endothelial cells. Due to its strong induction in tumors S100A4 is a promising target for cancer immunotherapy. By reverse immunology, using epitope prediction programs, we identified 3 HLA-A1-restricted peptide epitopes (S100A4 A1-1, A1-2, and A1-3) which are subject to human T cell responses as detected in peripheral blood of melanoma patients by means of IFN-gamma ELISPOT and cytotoxicity assays. In addition, IFN-gamma responses to S100A4 A1-2 can not only be induced by stimulation of T cells with peptide-loaded DC but also by stimulation with S100A4 protein-loaded DC, indicating that this epitope is indeed generated by processing of the endogenously expressed protein. In addition, S100A4 A1-2 reactive T cells demonstrate lysis of HLA-A1(+) fibroblasts in comparison to HLA-A1(-) fibroblasts. In summary, this HLA-A1-restricted peptide epitope is a candidate for immunotherapeutical approaches targeting S100A4-expressing cells in the tumor stroma.

Using the two color fluorescence (TCF) method, alloantibodies against subsets of T cells could be detected in sera from pregnant women with strong HLA antibodies. To preclude interference of these HLA antibodies with the recognition of the T cell antibodies, serum donors were selected which were HLA-Al, -B8, -DRw3. Their sera were tested on a panel of individuals homozygous for HLA-Al, -B8, -DRw3. By enriching peripheral mononuclear blood lymphocytes for Tgamma cells it could be shown that some of the sera reacted mainly with Tgamma and others with Tmu lymphocytes, while some sera reacted with both.

HLA-A, B, C and DR typing was performed in 21 unrelated healthy blood donors with selective IgA deficiency (< 0.02 G/l of IgA). A significant increase in HLA A1 (P < 0.05), HLA B8 (P < 0.01) and HLA DR3 (P < 0.001) was found. The frequency of HLA A28 was also increased (P < 0.05). Furthermore, HLA A28 was present in all four donors lacking DR3.

Smoking behavior has been associated in two independent European cohorts with the most common Caucasian human leukocyte antigen (HLA) haplotype (A1-B8-DR3). We aimed to test whether polymorphic members of the two odorant receptor (OR) clusters within the extended HLA complex might be responsible for the observed association, by genotyping a cohort of Hungarian women in which the mentioned association had been found. One hundred and eighty HLA haplotypes from Centre d'Etude du Polymorphisme Humain families were analyzed in silico to identify single-nucleotide polymorphisms (SNPs) within OR genes that are in linkage disequilibrium with the A1-B8-DR3 haplotype, as well as with two other haplotypes indirectly linked to smoking behavior. A nonsynonymous SNP within the OR12D3 gene (rs3749971T) was found to be linked to the A1-B8-DR3 haplotype. This polymorphism leads to a 97Thr→Ile exchange that affects a putative ligand binding region of the OR12D3 protein. Smoking was found to be associated in the Hungarian cohort with the rs3749971T allele (p=1.05×10-2), with higher significance than with A1-B8-DR3 (p=2.38×10-2). Our results link smoking to a distinct OR allele, and demonstrate that the rs3749971T polymorphism is associated with the HLA haplotype-dependent differential recognition of cigarette smoke components, at least among Caucasian women. © Copyright 2008, Mary Ann Liebert, Inc. 2008. Fil: Santos, Pablo Sandro Carvalho. Charité – Universitätsmedizin Berlin; Fil: Füst, George. Semmelweis Egyetem; Fil: Prohászka, Zoltán. Magyar Tudomanyos Akademia; . Semmelweis Egyetem; Fil: Volz, Armin. Charité – Universitätsmedizin Berlin; Fil: Horton, Roger. Wellcome Trust Sanger Institute; Fil: Miretti, Marcos Mateo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Universidad Nacional de Misiones. Instituto de Biología Subtropical; Argentina. Wellcome Trust Sanger Institute; Fil: Yu, Chack-Yung. Ohio State University; Estados Unidos Fil: Beck, Stephan G.. Ucl Cancer Institute; Fil: Uchanska-Ziegler, Barbara. Charité – Universitätsmedizin Berlin; Fil: Ziegler, Andreas. Charité – Universitätsmedizin Berlin

Acute enteroviral infections ranging from meningitis, pancreatitis to myocarditis are common and normally well controlled by the host immune system comprising virus-specific CD8+ cytotoxic T lymphocytes (CTL). However, in some patients enteroviruses and especially coxsackieviruses of group B are capable of inducing severe chronic forms of diseases such as chronic myocarditis. Currently, it is not known whether divergences in the CTL-related immune response may contribute to the different outcome and course of enterovirus myocarditis. A pre-requisite for the study of CTL reactions in patients with acute and chronic myocarditis is the identification of CTL epitopes. In order to define dominant enterovirus CTL epitopes, we have screened, by using gamma interferon (IFN-γ) ELISPOT, 62 HLA-A*01- and 59 HLA-A*02-positive healthy blood donors for pre-existing CTL reactions against 12 HLA-A*01 and 20 HLA-A*02 predicted CTL epitopes derived from coxsackieviruses of group B. Positive CTL reactions were verified by FACS analysis in a combined major histocompatibility complex-tetramer IFN-γ staining. A total of 14.8 % of all donors reacted against one of the three identified epitopes MLDGHLIAFDY, YGDDVIASY or GIIYIIYKL. The HLA-A*02-restricted epitope ILMNDQEVGV was recognized by 25 % of all tested blood donors. For this peptide, we could demonstrate specific granzyme B secretion, a strong cytolytic potential and endogenous processing. All four epitopes were homologous in 36–92 % of group B enteroviruses, providing a strong basis for monitoring the divergence of T-cell-based immune responses in enterovirus-induced acute and chronic diseases.

Tumor regressions have been observed in a small proportion of melanoma patients vaccinated with a MAGE-A3 peptide presented by HLA-A1, administered as peptide, ALVAC canarypox virus containing a MAGE-A3 minigene, or peptide-pulsed dendritic cells (DC). There was a correlation between tumor regression and the detection of anti–MAGE-3.A1 CTL responses. These responses were monoclonal and often of a very low magnitude after vaccination with peptide or ALVAC, and usually polyclonal and of a higher magnitude after DC vaccination. These results suggested that, at least in some patients, surprisingly few anti–MAGE-3.A1 T-cells could initiate a tumor regression process. To understand the role of these T cells, we carried out a functional analysis of anti–MAGE-3.A1 CTL clones derived from vaccinated patients who displayed tumor regression. The functional avidities of these CTL clones, evaluated in lysis assays, were surprisingly low, suggesting that high avidity was not part of the putative capability of these CTL to trigger tumor rejection. Most anti–MAGE-3.A1 CTL clones obtained after DC vaccination, but not after peptide or ALVAC vaccination, produced interleukin 10. Transcript profiling confirmed these results and indicated that approximately 20 genes, including CD40L, prostaglandin D2 synthase, granzyme K, and granzyme H, were highly differentially expressed between the anti–MAGE-3.A1 CTL clones derived from patients vaccinated with either peptide-ALVAC or peptide-pulsed DC. These results indicate that the modality of vaccination with a tumor-specific antigen influences the differentiation pathway of the antivaccine CD8 T-cells, which may have an effect on their capacity to trigger a tumor rejection response. [Cancer Res 2008;68(10):3931–40]

Major histocompatibility complex (MHC) class I multimer technology has become an indispensable immunological assay system to dissect antigen-specific cytotoxic CD8+T cell responses by flow cytometry. However, the development of high-throughput assay systems, in which T cell responses against a multitude of epitopes are analyzed, has been precluded by the fact that for each T cell epitope, a separatein vitroMHC refolding reaction is required. We have recently demonstrated that conditional ligands that disintegrate upon exposure to long-wavelength UV light can be designed for the human MHC molecule HLA-A2. To determine whether this peptide-exchange technology can be developed into a generally applicable approach for high throughput MHC based applications we set out to design conditional ligands for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Here, we describe the development and characterization of conditional ligands for this set of human MHC molecules and apply the peptide-exchange technology to identify melanoma-associated peptides that bind to HLA-A3 with high affinity. The conditional ligand technology developed here will allow high-throughput MHC-based analysis of cytotoxic T cell immunity in the vast majority of Western European individuals.

Adenovirus vectors have great potential in cancer gene therapy. Targeting of cancer-testis (CT) antigens, which are specifically presented at the surface of tumor cells by human leukocyte antigen (HLA) class I molecules, is an attractive option. In this study, a single-chain T-cell receptor (scTCR) directed against the CT antigen melanoma-associated antigen ( MAGE)-A1 in complex with the HLA class I molecule of haplotype HLA-A1 is fused with the C terminus of the adenovirus minor capsid protein IX. Propagation of a protein-IX (pIX)-gene-deleted human adenovirus 5 (HAdV-5) vector on cells that constitutively express the pIXscTCR fusion protein yielded viral particles with the pIXscTCR fusion protein incorporated in their capsid. Generated particles specifically transduced melanoma cell lines expressing the HLA-A1/MAGE-A1 target complex with at least 10-fold higher efficiency than control viruses. Whereas loading of HLA-A1-positive cells with MAGE-A1 peptides leads to enhanced transduction of the cells, the efficiency of virus transduction is strongly reduced if the HLA-A1 molecules are not accessible at the target cell. Taken together, these data provide proof of principle that pIXscTCR fusions can be used to target HAdV-5 vectors to tumor cells expressing intracellular CT antigens.

Background: Hemochromatosis is a recessively inherited disorder caused by a point mutation, C282Y of the HFE gene on chromosome 6p21.3 near the human leukocyte antigen (HLA) locus. It is unknown why some homozygotes develop a severe iron loading, while others do not. A recent study suggested that the A1-B8 haplotype may be associated with higher iron storage. Methods: We studied HLA haplotypes of 85 probands, 31 females and 54 males, and their family members from a rural population where A1-B8 was common. We tested the hypothesis of a modifying effect of the A1-B8 haplotype. Results: Most homozygotes had a mild phenotypic expression, and were often detected accidentally because of a laboratory routine including transferrin saturation. A disease-related morbidity [serum alanine aminotransferase (S-ALT) > 43 U] was present in 40%.Three had porphyria cutanea tarda. Two brothers with A1-B8 died of bronze diabetes, probably caused by co-inheritance of congenital spherocytosis. In females there were no significant differences in phenotypic expression between groups with regard to the presence or absence of A1-B8. Two females

Cancer cells' growth in three-dimensional (3D) architectures promotes resistance to drugs, cytokines, or irradiation. We investigated effects of 3D culture as compared to monolayers (2D) on melanoma cells' recognition by tumour-associated antigen (TAA)-specific HLA-A(*)0201-restricted cytotoxic T-lymphocytes (CTL). Culture of HBL, D10 (both HLA-A(*)0201+, TAA+) and NA8 (HLA-A(*)0201+, TAA-) melanoma cells on polyHEMA-coated plates, resulted in generation of 3D multicellular tumour spheroids (MCTS). Interferon-gamma (IFN-gamma) production by HLA-A(*)0201-restricted Melan-A/MART-1(27-35) or gp 100(280-288)-specific CTL clones served as immunorecognition marker. Co-culture with melanoma MCTS, resulted in defective TAA recognition by CTL as compared to 2D as witnessed by decreased IFN-gamma production and decreased Fas Ligand, perforin and granzyme B gene expression. A multiplicity of mechanisms were potentially involved. First, MCTS per se limit CTL capacity of recognising HLA class I restricted antigens by reducing exposed cell surfaces. Second, expression of melanoma differentiation antigens is downregulated in MCTS. Third, expression of HLA class I molecules can be downregulated in melanoma MCTS, possibly due to decreased interferon-regulating factor-1 gene expression. Fourth, lactic acid production is increased in MCTS, as compared to 2D. These data suggest that melanoma cells growing in 3D, even in the absence of immune selection, feature characteristics capable of dramatically inhibiting TAA recognition by specific CTL.