Search

Background and aims: genetic predisposition to autoimmune hepatitis (AIH) in adults is associated with possession of human leukocyte antigen (HLA) class I (A*01, B*08) and class II (DRB1*03, -04, -07, or -13) alleles, depending on geographic region. Juvenile autoimmune liver disease (AILD) comprises AIH-1, AIH-2, and autoimmune sclerosing cholangitis (ASC), which are phenotypically different from their adult counterparts. We aimed to define the relationship between HLA profile and disease course, severity, and outcome in juvenile AILD. Approach and results: we studied 236 children of European ancestry (152 female [64%], median age 11.15 years, range 0.8-17), including 100 with AIH-1, 59 with AIH-2, and 77 with ASC. The follow-up period was from 1977 to June 2019 (median 14.5 years). Class I and II HLA genotyping was performed using PCR/sequence-specific primers. HLA B*08, -DRB1*03, and the A1-B8-DR3 haplotype impart predisposition to all three forms of AILD. Homozygosity for DRB1*03 represented the strongest risk factor (8.8). HLA DRB1*04, which independently confers susceptibility to AIH in adults, was infrequent in AIH-1 and ASC, suggesting protection; and DRB1*15 (DR15) was protective against all forms of AILD. Distinct HLA class II alleles predispose to the different subgroups of juvenile AILD: DRB1*03 to AIH-1, DRB1*13 to ASC, and DRB1*07 to AIH-2. Possession of homozygous DRB1*03 or of DRB1*13 is associated with fibrosis at disease onset, and possession of these two genes in addition to DRB1*07 is associated with a more severe disease in all three subgroups. Conclusions: unique HLA profiles are seen in each subgroup of juvenile AILD. HLA genotype might be useful in predicting responsiveness to immunosuppressive treatment and course., Fifth Medical Center of PLA General Hospital; Roger Dobson Funds; King's College Hospital Charity; Medical Research Council Clinician Scientist Fellowship; Medical Research Council PhD Studentship; Alex Mowat PhD Studentship; King's College Hospital Charity; National Institute for Health Research University College London Hospital/University College London Biomedical Research Centre

BackgroundHuman Papillomavirus type 18 (HPV18) is a high-risk HPV that is commonly associated with cervical cancer. HPV18 oncogenes E6 and E7 are associated with the malignant transformation of cells, thus the identification of human leukocyte antigen (HLA)-restricted E6/E7 peptide-specific CD8 + T cell epitopes and the creation of a HPV18 E6/E7 expressing cervicovaginal tumor in HLA-A2 transgenic mice will be significant for vaccine development.MethodsIn the below study, we characterized various human HLA class I-restricted HPV18 E6 and E7-specific CD8 + T cells mediated immune responses in HLA class I transgenic mice using DNA vaccines encoding HPV18E6 and HPV18E7. We then confirmed HLA-restricted E6/E7 specific CD8 + T cell epitopes using splenocytes from vaccinated mice stimulated with HPV18E6/E7 peptides. Furthermore, we used oncogenic DNA plasmids encoding HPV18E7E6(delD70), luciferase, cMyc, and AKT to create a spontaneous cervicovaginal carcinoma model in HLA-A2 transgenic mice.ResultsTherapeutic HPV18 E7 DNA vaccination did not elicit any significant CD8 + T cell response in HLA-A1, HLA-24, HLA-B7, HLA-B44 transgenic or wild type C57BL/6 mice, but it did generate a strong HLA-A2 and HLA-A11 restricted HPV18E7-specific CD8 + T cell immune response. We found that a single deletion of aspartic acid (D) at location 70 in HPV18E6 DNA abolishes the presentation of HPV18 E6 peptide (aa67-75) by murine MHC class I. We found that the DNA vaccine with this mutant HPV18 E6 generated E6-specific CD8 + T cells in HLA-A2. HLA-A11, HLA-A24 and HLA-b40 transgenic mice. Of note, HLA-A2 restricted, HPV18 E7 peptide (aa7-15)- and HPV18 E6 peptide (aa97-105)-specific epitopes are endogenously processed by HPV18 positive Hela-AAD (HLA-A*0201/Dd) cells. Finally, we found that injection of DNA plasmids encoding HPV18E7E6(delD70), AKT, cMyc, and SB100 can result in the development of adenosquamous carcinoma in the cervicovaginal tract of HLA-A2 transgenic mice.ConclusionsWe characterized various human HLA class I-restricted HPV18 E6/E7 peptide specific CD8 + T cell epitopes in human HLA class I transgenic mice. We demonstrated that HPV18 positive Hela cells expressing chimeric HLA-A2 (AAD) do present both HLA-A2-restricted HPV18 E7 (aa7-15)- and HPV18 E6 (aa97-105)-specific CD8 + T cell epitopes. A mutant HPV18E6 that had a single deletion at location 70 obliterates the E6 presentation by murine MHC class I and remains oncogenic. The identification of these human MHC restricted HPV antigen specific epitopes as well as the HPV18E6/E7 expressing adenosquamous cell carcinoma model may have significant future translational potential.

Cytotoxic T lymphocyte (CTL)-based cancer immunotherapies have shown great promise for inducing clinical regressions by targeting tumor-associated antigens (TAA). To expand the TAA landscape of pancreatic ductal adenocarcinoma (PDAC), we performed tandem mass spectrometry analysis of HLA class I-bound peptides from 35 PDAC patient tumors. This identified a shared HLA-A*0101 restricted peptide derived from co-transcriptional activator Vestigial-like 1 (VGLL1) as a putative TAA demonstrating overexpression in multiple tumor types and low or absent expression in essential normal tissues. Here we show that VGLL1-specific CTLs expanded from the blood of a PDAC patient could recognize and kill in an antigen-specific manner a majority of HLA-A*0101 allogeneic tumor cell lines derived not only from PDAC, but also bladder, ovarian, gastric, lung, and basal-like breast cancers. Gene expression profiling reveals VGLL1 as a member of a unique group of cancer-placenta antigens (CPA) that may constitute immunotherapeutic targets for patients with multiple cancer types., Cytotoxic T lymphocyte (CTL)-based immunotherapies can induce tumor regressions by targeting HLA class I-bound tumor-associated peptides. Here, the authors identified a peptide derived from Vestigial-like 1 (VGLL1) as a shared, potentially therapeutic CTL target expressed by multiple cancer types.

To verify whether PsA-associated HLA alleles proposed in other populations are also related to PsA in Chinese Han population, a study of PsA susceptible alleles in the HLA-A, HLA-B, HLA-C and HLA-DRB1 alleles was presented for Chinese Han population. Genotyping was performed by Illumina Miseq platform (Illumina, USA). 50 subtypes and 77 subtypes of HLA-A, HLA-B, HLA-C and HLA-DRB1 with minor allele frequency (MAF) > 1% were genotyped from two-digit and four-digit resolution analysis in 111 PsA and 207 HCs (healthy controls) collected from Chinese Han population, respectively. Data handling, quality control and association analysis were performed using SPSS 25.0 software. In risk estimate, by mean of Bonferroni correction, a newfound four-digit allele HLA-A*01:01 [P = 5.5 × 10−4, OR 3.35 (1.69–6.66)], four-digit allele HLA-C*06:02 [P = 8.5 × 10−7, OR 3.80 (2.23–6.47)] and six two-digit alleles HLA-A*01 [P = 5.2 × 10−5, OR 3.43 (1.89–6.23)], HLA-B*13 [P = 4.0 × 10−6, OR 2.65 (1.76–4.01)], HLA-B*27 [P = 7.5 × 10−4, OR 5.84 (2.09–16.29)], HLA-B*57 [P = 5.8 × 10−5, OR 20.10 (4.65–86.83)], HLA-C*03 [P = 2.1 × 10−4, OR 0.40 (0.25–0.65)], HLA-C*06 [P = 1.9 × 10−12, OR 4.48 (2.95–6.81)] showed statistical significance by the univariate binary logistic regression analysis. Besides, in the binary logistic regression analysis with multiple variables, when the two alleles HLA-A*01:01 and HLA-C*06:02 were considered as covariates, HLA-A*01:01 [P = 2.7 × 10−3,OR 2.95 (1.46–5.98)] also showed significant association for PsA as risk factor, but may be not the main risk factor [HLA-C*06:02, P = 3.0 × 10−6, OR 3.68 (2.13–6.37)]. When all the above two-digit alleles were included as covariates, HLA-A*01 [P = 4.8 × 10−2, OR 2.00 (1.01–3.94)], HLA-B*13 [P = 4.2 × 10−5, OR 2.62 (1.65–4.16)], HLA-B*27 [P = 1.7 × 10−4, OR 7.62 (2.64–21.96)], HLA-B*57 [P = 2.97 × 10−4, OR 15.90 (3.55–71.18)], HLA-C*06 [P = 6.1 × 10−5, OR 2.70 (1.66–4.40)] showed significant for PsA as risk factors, HLA-C*03 [OR 0.65 (0.39–1.09), P = 0.10] showed no association with PsA. In conclusion, we assessed HLA-A, HLA-B, HLA-C and HLA-DRB1 alleles in PsA cohort of Chinese Han population, found HLA-A*01:01 and HLA-A*01 may be the susceptible genes associated with PsA, and also confirmed the association of four loci with PsA in Chinese Han population. These findings may extend the susceptibility HLA alleles of PsA and help in developing possible genetic markers to predict PsA.

The intracellular pathway of cross-presentation, which allows MHC class I–restricted presentation of peptides derived from exogenous Ags, remains poorly defined and may vary with the nature of the exogenous Ag and the type of APC. It can be cytosolic, characterized by proteasome and TAP dependency, or vacuolar, usually believed to be proteasome and TAP independent. Cross-presentation is particularly effective with long synthetic peptides, and we previously reported that the HLA-A2–restricted cross-presentation of a long peptide derived from melanoma Ag gp100 by human monocyte-derived immature dendritic cells occurred in a vacuolar pathway, making use of newly synthesized HLA-A2 molecules that follow a nonclassical secretion route. In this article, we show that the HLA-A1–restricted cross-presentation of a long peptide derived from tumor Ag MAGE-A3 by human monocyte-derived immature dendritic cells also follows a vacuolar pathway. However, as opposed to the HLA-A2–restricted peptide, cross-presentation of the HLA-A1–restricted peptide is TAP dependent. We show that this paradoxical TAP-dependency is indirect and reflects the need for TAP to load HLA-A1 molecules with peptides in the endoplasmic reticulum, to allow them to escape the endoplasmic reticulum and reach the vacuole, where peptide exchange with the cross-presented peptide likely occurs. Our results confirm and extend the involvement of the vacuolar pathway in the cross-presentation of long peptides, and indicate that TAP-dependency can no longer be used as a key criterion to distinguish the cytosolic from the vacuolar pathway of cross-presentation. They also stress the existence of an alternative secretory route for MHC class I, which will be worthy of further studies.

Adenoviruses are a major cause of infectious mortality in children following allogeneic hematopoietic stem cell transplantation, with adoptive transfer of adenovirus-specific T cells being an effective therapeutic approach. We have previously shown that T cells specific for the peptide epitope LTDLGQNLLY were protective. In this study, we aimed to establish a viral dissemination assay to measure the antiviral capacity of T cells specific for this and other peptide epitopes in an infectious setting. We used replication-competent adenovirus 11 (Ad11pGFP) and adenovirus 5 containing adenovirus 35 fiber (Ad5F35GFP) viruses and T cells specific for HLA-A*01–restricted LTDLGQNLLY, HLA-B*07–restricted KPYSGTAYNAL, and HLA-A*02–restricted LLDQLIEEV peptide epitopes. T cells in PBMC from healthy donors were expanded with peptide and IL-2 or treated with IL-2 alone to serve as nonstimulated control cells, and then these expanded or nonstimulated CD8+ cells were purified and cocultured with autologous monocytes infected with adenovirus at low multiplicity of infection. After 3 d, the number of infected GFP+ monocytes and, hence, viral dissemination was quantified by flow cytometry. T cells expanded with LTDLGQNLLY peptide from multiple HLA-A*01+ donors prevented adenovirus dissemination, and nonstimulated T cells did not prevent dissemination, thus, indicating that LTDLGQNLLY-specific T cells have high antiviral capacity. Similarly, expanded KPYSGTAYNAL- and LLDQLIEEV-specific T cells could prevent viral dissemination. However, the frequency of expanded T cells specific for these last two epitopes was variable between donors with consequent variable prevention of adenoviral dissemination. Taken together, we demonstrate that T cells specific for three peptide epitopes, from both structural and nonstructural proteins, can prevent adenoviral dissemination and provide a novel method to measure the antiviral capacity of adenovirus-specific T cell responses.

Cell surface antigen discovery is of great interest for biomedical research both for isolation of rare cell populations and therapeutic targeting. We developed a rapid, cost-effective, fully in vitro technology which facilities the simultaneous target discovery and human antibody generation on the surface of virtually any cell population of interest. We apply our technique to human colorectal cancer-initiating cells (CICs) and identify hundreds of unique human antibodies. We characterized the top three antibody candidates targeting these CICs and identify their protein targets as integrin α7 (ITGA7), HLA-A1 and integrin β6 (ITGB6). We demonstrate that these antibodies can be used to isolate self-renewing colorectal CICs, and that the integrin α7 antibody can prospectively identify glioblastoma brain tumor initiating cells as well as human muscle stem cells. We also demonstrate that genetic ablation of integrin β6 impedes colorectal CIC function. The methodology can be readily applied to other cell populations including stem cells, cancer, or immune cells to facilitate the rapid identification of novel targets and simultaneous generation of potent and specific antibodies with therapeutic potential.

Background Heterozygosity at HLA class I loci is generally considered beneficial for host defense. We report here an element of HLA class I homozygosity that may or may not help preserve its existence in populations but which could indicate a new avenue for antiviral research. Methods Lymphocytes from serologically HLA-homozygous or -heterozygous donors were examined for synthesis of influenza virus proteins and RNA after exposure to virus as peripheral blood mononuclear cells. The virus-exposed lymphocytes were also examined for internalization of the virus after exposure, and for susceptibility to virus-specific cytotoxic T lymphocytes in comparison with virus-exposed monocytes/macrophages and unseparated peripheral blood mononuclear cells. Results were compared using two-tailed Fisher’s exact test. Results Serologically-defined HLA-A2-homozygous lymphocytes, in contrast to heterozygous lymphocytes, did not synthesize detectable influenza virus RNA or protein after exposure to the virus. HLA-A2-homozygous lymphocytes, including both homozygous and heterozygous donors by genetic sequence subtyping, did internalize infectious virus but were not susceptible to lysis by autologous virus-specific cytotoxic T lymphocytes (“fratricide”). Similar intrinsic resistance to influenza virus infection was observed with HLA-A1- and HLA-A11-homozygous lymphocytes and with HLA-B-homozygous lymphocytes. Conclusions A significant proportion of individuals within a population that is characterized by common expression of HLA class I alleles may possess lymphocytes that are not susceptible to influenza virus infection and thus to mutual virus-specific lysis. Further study may identify new approaches to limit influenza virus infection.

Human leukocyte antigen (HLA) class I presentation pathway plays a central role in natural killer (NK) cell and cytotoxic T-cell activities against BK polyomavirus (BKPyV) DNAemia. We determined the risk of sustained BKPyV DNAemia in 175 consecutive renal transplant recipients considering the simultaneous effect of donor/recipient HLA class I antigens and pre- or post-transplant variables. Median (IQR) age was 53 (44-64) years, and 37% of patients were female. 40 patients (22.9%) developed sustained BKPyV DNAemia [median (IQR) viral load: 9740 (4350-17 125) copies/ml]. In the Cox proportional hazard analysis, HLA-A1 (HR: 3.06, 95% CI: 1.51-6.17) and HLA-B35-Cw4 (HR: 4.63, 95% CI: 2.12-10.14) significantly increased the risk of sustained BKPyV DNAemia, while 2 HLA-C mismatches provided a marginally protective effect (HR: 0.32, 95% CI: 0.10-0.98). HLA-Cw4 is a ligand for NK cell inhibitory receptor, and HLA-B35 is in strong linkage disequilibrium with the HLA-Cw4 allele. The association between HLA-B35-Cw4 expression and sustained BKPyV DNAemia supports the important role of cytotoxic T cells and NK cells that would normally control BKPyV activation through engagement with immunoglobulin-like killer receptors (KIRs). Further studies are required to investigate the effect of HLA-C alleles along with NK cell activity against BKPyV DNAemia.

Objectives Asthma is one of the most common major noncommunicable diseases in the world and affects individuals of all ages. Medication is used to achieve and maintain quality of life (QOL) for people with asthma. Telehealth interventions offer optimized and personalized symptom monitoring with timely treatment adjustment and the potential to increase medication adherence for individuals with asthma. This study examines and synthesizes the available data on the change in the QOL for patients with asthma who use interactive telehealth interventions, and identifies the most effective telehealth modalities used for intervention in this area. Methods Literature searches were conducted in 5 databases in November 2018 for studies measuring a change in QOL for patients with asthma. Study QOL outcomes, where possible, were pooled in a meta-analysis. Results Seventeen publications (describing 16 studies) comprising 2015 patients were included. Based on a meta-analysis, interactive telehealth interventions can improve QOL outcomes for people living with asthma, although the improved effects may be small: web portals (0.51, 95% confidence interval [CI] –0.00 to 1.03), interactive smartphone apps (0.30, 95% CI –0.16 to 0.76) and remote monitoring (standardized mean difference 0.20, 95% CI –0.11 to 0.52). Intervention delivery modalities identified include interactive web portals, smartphone apps, and remote monitoring programs. Conclusions The findings provide a comprehensive overview of the available literature on interactive telehealth interventions, including interactive web portals, smartphone apps, and remote monitoring programs. These findings demonstrated that a positive change in QOL can be attributed to these interventions and provide evidence for the implementation of telehealth interventions for individuals with asthma.

Specificity analyses of peptide binding to human leukocyte antigen (HLA)-A molecules have been hampered due to a lack of proper monoclonal antibodies (mAbs) for certain allomorphs, such as the prevalent HLA-A1 for Caucasians and HLA-A11 for Asians. We developed a mAb that recognizes a conformational epitope common to most HLA-A allomorphs. The mAb, named A-1, does not discriminate peptides by amino acid sequences, making it suitable for measuring peptide binding. A stabilization assay using TAP-deficient cell lines and A-1 was developed to investigate the specificity of peptide binding to HLA-A molecules. Regarding the evolution of HLA-A genes, the A-1 epitope has been conserved among most HLA-A allomorphs but was lost when the HLA-A gene diversified into the HLA-A*32, HLA-A*31, and HLA-A*33 lineages together with HLA-A*29 after bifurcating from the HLA-A*25 and HLA-A*26 branchs. The establishment of A-1 is expected to help researchers investigate the peptide repertoire and develop computational tools to identify cognate peptides. Since no HLA-A locus-specific mAb has been available, A-1 will also be useful for analyzing the locus-specific regulation of the HLA gene expression.

Background aims Herpes simplex virus (HSV) reactivation and infection is common in patients undergoing hematopoietic stem cell transplant (HSCT) and requires routine antiviral prophylaxis. Drug-resistant strains are increasingly common, and effective alternative therapy is currently unavailable. We generated and characterized HSV-1-specific T cells for use in adoptive cellular immunotherapy following allogeneic stem cell transplantation. Methods Peripheral blood mononuclear cells from HLA-A1 and HLA-A2 HSV-seropositive hereditary hemochromatosis donors were used as the antigen source. Three HLA-A1 and four HLA-A2 specific epitopes were used for stimulation of T cells. Cells were stimulated with antigen-pulsed dendritic cells and cultured for 21 days in medium with interleukin (IL)-2. Cultured cells were phenotyped and tested for cytokine production, proliferation and cytotoxicity. Results There was a 5.3-fold expansion in total cell numbers over 21 days of culture, with 35% of T cells being CD8 positive. Thirty-five percent, 21% and 5% of CD8 cells secreted interferon-γ, tumor necrosis factor-α and IL-2 upon HSV antigen re-stimulation. More than 50% of antigen-specific T cells secreted multiple cytokines. Cultured T cells proliferated upon antigen re-stimulation and lysed HSV-1 peptide and virus-infected targets. Conclusions It is feasible to generate functional HSV-1 specific T cells from the blood of HLA-A1 and HLA-A2 HSV-seropositive donors using specific peptides. The utility of these cells in preventing and treating HSV-1 reactivation in allogeneic HSCT will need to be tested clinically.

Purpose Melanoma is the most aggressive form of skin cancer. Chemotherapy at a late stage fails due to low accumulation in tumors, indicating the need for targeted therapy. Materials and methods To increase drug uptake by tumor cells, we have targeted doxorubicin-containing liposomes using a T-cell receptor (TCR)-like antibody (scFv G8 and Hyb3) directed against melanoma antigen A1 (MAGE-A1) presented by human leukocyte antigen A1 (M1/A1). With the use of flow cytometry and confocal microscopy, we have tested our formulation in vitro. In vivo pharmacokinetics was done in tumor-free nu/nu mice, while biodistribution and efficacy study was done in nu/nu mice xenograft. Results We demonstrated two to five times higher binding and internalization of these immunoliposomes by M1+/A1+ melanoma cells in vitro in comparison with nontargeted liposomes. Cytotoxicity assay showed significant tumor cell kill at 10 µM doxorubicin (DXR) for targeted vs nontargeted liposomes. In vivo pharmacokinetics of nontargeted and targeted liposomes were similar, while accumulation of targeted liposomes was 2- to 2.5-fold and 6.6-fold enhanced when compared with nontargeted liposomes and free drug, respectively. Notably, we showed a superior antitumor activity of MAGE-A1-targeted DXR liposomes toward M1+/A1+ expressing tumors in mice compared with the treatment of M1−/A1+ tumors. Our results indicate that targeted liposomes showed better cytotoxicity in vitro and pharmacokinetics in vivo. Conclusion Liposomes decorated with TCR-mimicking scFv antibodies effectively and selectively target antigen-positive melanoma. We showed that DXR-loaded liposomes coupled to anti-M1/-A1 scFv inflict a significant antitumor response. Targeting tumor cells specifically promotes internalization of drug-containing nanoparticles and may improve drug delivery and ultimately antitumor efficacy. Our data argue that targeting MAGE in A1 context, by nanosized carriers decorated with TCR-like antibodies mimicking scFv, can be used as a theragnostic platform for drug delivery, immunotherapy, and potentially imaging, and diagnosis of melanoma.

Newly-emerged and vaccine-mismatched influenza A viruses (IAVs) result in a rapid global spread of the virus due to minimal antibody-mediated immunity. In that case, established CD8+ T-cells can reduce disease severity. However, as mutations occur sporadically within immunogenic IAV-derived T-cell peptides, understanding of T-cell receptor (TCRαβ) cross-reactivity towards IAV variants is needed for a vaccine design. Here, we investigate TCRαβ cross-strain recognition across IAV variants within two immunodominant human IAV-specific CD8+ T-cell epitopes, HLA-B*37:01-restricted NP338-346 (B37-NP338) and HLA-A*01:01-restricted NP44-52 (A1-NP44). We find high abundance of cross-reactive TCRαβ clonotypes recognizing distinct IAV variants. Structures of the wild-type and variant peptides revealed preserved conformation of the bound peptides. Structures of a cross-reactive TCR-HLA-B37-NP338 complex suggest that the conserved conformation of the variants underpins TCR cross-reactivity. Overall, cross-reactive CD8+ T-cell responses, underpinned by conserved epitope structure, facilitates recognition of distinct IAV variants, thus CD8+ T-cell-targeted vaccines could provide protection across different IAV strains., Mutations within immunological epitope containing regions of influenza A virus can impair the established immune response between influenza strains and could impact rational vaccine design. Here Grant et al. examine the presence, structural impact and cross reactivity of two human immunodominant influenza epitope variants.

The susceptibility to Epstein-Barr virus (EBV)-related post-transplant lymphoproliferative disorder (PTLD) may be affected by the human leukocyte antigen (HLA) type. We investigated HLA-A and HLA-B allele frequencies, focusing on HLA-A1 and -A2, in a population-based case series of EBV + (n = 60) and EBV- (n = 44) PTLD after solid organ transplantation. The proportion of EBV + PTLD was highest in HLA-A1 homozygotes (100%), lower in carriers of HLA-A1/AX (79%), HLA-A1/A2 (55%), HLA-A2/AX (54%), and lowest in HLA-A2 homozygotes (37%). HLA-A1 type was overrepresented (22% versus 7%, p = 0.05) and HLA-A2 type underrepresented (57% versus 80%, p = 0.01) in patients with EBV + compared with EBV - PTLD. EBV + PTLD in HLA-A1 carriers developed almost exclusively in already EBV-seropositive individuals. EBV status of PTLD was not related to any other HLA-A or HLA-B type. Our findings suggest that HLA-A1 carriers may have an increased risk of EBV + PTLD due to a decreased ability to control the latent EBV infection.

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

Summary The recent HIV-1 vaccine failures highlight the need to better understand virus-host interactions. One key question is why CD8+ T cell responses to two HIV-Gag regions are uniquely associated with delayed disease progression only in patients expressing a few rare HLA class I variants when these regions encode epitopes presented by ∼30 more common HLA variants. By combining epitope processing and computational analyses of the two HIV subtypes responsible for ∼60% of worldwide infections, we identified a hitherto unrecognized adaptation to the antigen-processing machinery through substitutions at subtype-specific motifs. Multiple HLA variants presenting epitopes situated next to a given subtype-specific motif drive selection at this subtype-specific position, and epitope abundances correlate inversely with the HLA frequency distribution in affected populations. This adaptation reflects the sum of intrapatient adaptations, is predictable, facilitates viral subtype diversification, and increases global HIV diversity. Because low epitope abundance is associated with infrequent and weak T cell responses, this most likely results in both population-level immune evasion and inadequate responses in most people vaccinated with natural HIV-1 sequence constructs. Our results suggest that artificial sequence modifications at subtype-specific positions in vitro could refocus and reverse the poor immunogenicity of HIV proteins., Graphical Abstract, Highlights • HLA class I variants drive selection pressures on Gag to limit epitope production • The strength of the selective pressure is positively correlated to HLA frequencies • HIV adaptation to limit epitope production occurs at subtype-specific HIV motifs • HIV adapts in a predictable way to HLA frequencies in newly infected populations, CD8+ T cell responses against HIV-1 effectively delay disease progression in a minority of patients with relatively rare HLA variants but are ineffective in most. Here, Tenzer et al. identify fundamental HIV-1 adaptation to the conserved human antigen-processing machinery that feeds epitopes to HLA. This adaptation occurs at subtype-specific motifs, facilitates subtype diversification, is predictable, and results in CD8 epitope abundances that correlate inversely with the HLA allele frequencies in affected populations. Thus, HIV vaccine immunogenicity might be increased by unnatural substitutions at subtype-specific motifs.

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.

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]