Phenotyping of 60 cultured human gliomas and 34 other neuroectodermal tumors by means of monoclonal antibodies against glioma, melanoma and HLA-DR antigens.

The reactivity spectrum of three monoclonal antibodies (Mabs) to human malignant glioma, five Mabs to melanomas and one Mab anti-HLA-DR was investigated by an indirect antibody binding radioimmunoassay on a panel of cells derived from 60 glioma lines, including 47 malignant astrocytomas, 11 low-grade astrocytomas and two malignant ependymomas as well on cells from 12 melanoma, three neuroblastoma, three medulloblastoma, two schwannoma, two retinoblastoma, two choroïd plexus papilloma, ten meningioma and 12 unrelated tumor lines. The anti-glioma Mabs BF7 and GE2 reacted preferentially with gliomas, while the anti-glioma Mab CG12 reacted with gliomas, melanomas, neuroblastomas and medulloblastomas. The five anti-melanoma Mabs reacted with gliomas, neuroblastomas and medulloblastomas. The anti-HLA-DR Mab D1-12 reacted with gliomas, melanomas and some meningiomas. On the basis of the data presented, we describe three different antigenic systems; the first one is glioma-associated, the second one is related to differentiation antigens expressed on cells derived from the neuroectoderm and the third is represented by HLA-DR antigens which are expressed not only on B-lymphoblastoid cells but also on melanomas and gliomas.

Final antigenic Melan-A peptides produced directly by the proteasomes are preferentially selected for presentation by HLA-A*0201 in melanoma cells.

The melanoma-associated protein Melan-A contains the immunodominant CTL epitope Melan-A(26/27-35)/HLA-A*0201 against which a high frequency of T lymphocytes has been detected in many melanoma patients. In this study we show that the in vitro degradation of a polypeptide encompassing Melan-A(26/27-35) by proteasomes produces both the final antigenic peptide and N-terminally extended intermediates. When human melanoma cells expressing the corresponding fragments were exposed to specific CTL, those expressing the minimal antigenic sequence were recognized more efficiently than those expressing the N-terminally extended intermediates. Using a tumor-reactive CTL clone, we confirmed that the recognition of melanoma cells expressing an N-terminally extended intermediate of Melan-A is inefficient. We demonstrated that the inefficient cytosolic trimming of N-terminally extended intermediates could offer a selective advantage for the preferred presentation of Melan-A peptides directly produced by the proteasomes. These results imply that both the proteasomes and postproteasomal peptidases limit the availability of antigenic peptides and that the efficiency of presentation may be affected by conditions that alter the ratio between fully and partially processed proteasomal products.

Genome-wide association study identifies new HLA class II haplotypes strongly protective against narcolepsy.

Narcolepsy is a rare sleep disorder with the strongest human leukocyte antigen (HLA) association ever reported. Since the associated HLA-DRB1*1501-DQB1*0602 haplotype is common in the general population (15-25%), it has been suggested that it is almost necessary but not sufficient for developing narcolepsy. To further define the genetic basis of narcolepsy risk, we performed a genome-wide association study (GWAS) in 562 European individuals with narcolepsy (cases) and 702 ethnically matched controls, with independent replication in 370 cases and 495 controls, all heterozygous for DRB1*1501-DQB1*0602. We found association with a protective variant near HLA-DQA2 (rs2858884; P < 3 x 10(-8)). Further analysis revealed that rs2858884 is strongly linked to DRB1*03-DQB1*02 (P < 4 x 10(-43)) and DRB1*1301-DQB1*0603 (P < 3 x 10(-7)). Cases almost never carried a trans DRB1*1301-DQB1*0603 haplotype (odds ratio = 0.02; P < 6 x 10(-14)). This unexpected protective HLA haplotype suggests a virtually causal involvement of the HLA region in narcolepsy susceptibility.

Prediction of cross-recognition of peptide-HLA A2 by Melan-A-specific cytotoxic T lymphocytes using three-dimensional quantitative structure-activity relationships.

The cross-recognition of peptides by cytotoxic T lymphocytes is a key element in immunology and in particular in peptide based immunotherapy. Here we develop three-dimensional (3D) quantitative structure-activity relationships (QSARs) to predict cross-recognition by Melan-A-specific cytotoxic T lymphocytes of peptides bound to HLA A*0201 (hereafter referred to as HLA A2). First, we predict the structure of a set of self- and pathogen-derived peptides bound to HLA A2 using a previously developed ab initio structure prediction approach [Fagerberg et al., J. Mol. Biol., 521-46 (2006)]. Second, shape and electrostatic energy calculations are performed on a 3D grid to produce similarity matrices which are combined with a genetic neural network method [So et al., J. Med. Chem., 4347-59 (1997)] to generate 3D-QSAR models. The models are extensively validated using several different approaches. During the model generation, the leave-one-out cross-validated correlation coefficient (q (2)) is used as the fitness criterion and all obtained models are evaluated based on their q (2) values. Moreover, the best model obtained for a partitioned data set is evaluated by its correlation coefficient (r = 0.92 for the external test set). The physical relevance of all models is tested using a functional dependence analysis and the robustness of the models obtained for the entire data set is confirmed using y-randomization. Finally, the validated models are tested for their utility in the setting of rational peptide design: their ability to discriminate between peptides that only contain side chain substitutions in a single secondary anchor position is evaluated. In addition, the predicted cross-recognition of the mono-substituted peptides is confirmed experimentally in chromium-release assays. These results underline the utility of 3D-QSARs in peptide mimetic design and suggest that the properties of the unbound epitope are sufficient to capture most of the information to determine the cross-recognition.

Does Influenza Vaccination Induce De Novo HLA Alloantibody Formation in Transplant Recipients?

Introduction: Infl uenza vaccination is recommended for all solid organ transplant recipients. However, some centers are reluctant to give annual vaccination due to concerns about precipitating rejection. A proposed mechanism of this is vaccineinduced development of cellular and humoral responses to donor HLA antigens. We studied the induction of HLA Ab in a cohort of lung transplant recipients receiving infl uenza vaccination. Methods: Adult lung transplant recipients were immunized with 0.5 mL intramuscular seasonal infl uenza vaccine followed by 0.1 mL intradermal booster at 4 weeks as part of a previous study. Sera were collected pre-vaccination and at 4, 8 weeks post-vaccination. Post-vaccination sera were analyzed for HLA Ab using fl owPRA specifi c beads (One Lambda Inc). A positive result was defi ned as 5%. Positive samples were further analyzed for antibody specifi city by single antigen bead testing. Pre-vaccination sera were tested only only if post-vaccination sample screen was positive for HLA Ab. The presence of HLA Ab was correlated to vaccine seroresponse and rejection episodes. Results: Sixty patients were included with equal numbers of men and women. Mean age of patients was 47.3 years (range 20.7-72.4). Median time post-transplant was 1.3 years (range 85 days - 17 years). One patient was excluded due to an uninterpretable baseline screen result. 16/59 (27.1%) patients were positive for HLA Ab both in both pre- and post-vaccination samples. Of these, 12/16 (75%) had antibody against HLA Class I (majority A30,A31,B27,B44), 2/16 (12.5%) had antibody against HLA class II (majority DQ4, DQ7), and 2/16 (12.5%) had antibody against both Class I & II. There was no signifi cant increase in existing HLA Ab post-vaccination. Of the 16 patients, only one (6.3%) patient had de novo HLA Ab and this was determined to be non donor specifi c. Factors such as gender, time from transplant, immunosuppression, and acute rejection episodes did not correlate with presence of HLA Ab. HLA Ab was not associated with seroconversion to to vaccine antigens. Conclusions: Our data support that receiving the annual infl uenza vaccine does not lead to the generation of de novo donor specifi c antibodies in lung transplant recipients or upregulation of existing HLA Ab.

Recognition of major histoincompatibilities after transplantation with marrow from HLA closely matched donors.

BACKGROUND: To determine the extent to which major histoincompatibilities are recognized after bone marrow transplantation, we characterized the specificity of the cytotoxic T lymphocytes isolated during graft-versus-host disease. We studied three patients transplanted with marrow from donors who were histoincompatible for different types of HLA antigens. METHODS: Patient 1 was mismatched for one "ABDR-antigen" (HLA-A2 versus A3). Two patients were mismatched for antigens that would usually not be taken into account by standard selection procedures: patient 2 was mismatched for an "HLA-A subtype" (A*0213 versus A*0201), whereas patient 3 was mismatched for HLA-C (HLA-C*0501 versus HLA-C*0701). All three HLA class I mismatches were detected by a pretransplant cytotoxic precursor test. RESULTS: Analysis of the specificity of the cytotoxic T lymphocyte clones isolated after transplantation showed that the incompatibilities detected by the pretransplant cytotoxic precursor assay were the targets recognized during graft-versus-host disease. CONCLUSIONS: Independent of whether the incompatibility consisted of a "full" mismatch, a "subtype" mismatch, or an HLA-C mismatch, all clones recognized the incompatible HLA molecule. In addition, some of these clones had undergone antigen selection and were clearly of higher specificity than the ones established before transplantation, indicating that they had been participating directly in the antihost immune response.

An epidemiologic study to determine the prevalence of the HLA-B*5701 allele among HIV-positive patients in Europe.

OBJECTIVES: HLA-B*5701 is a major histocompatibility complex class I allele associated with an immunologically-mediated hypersensitivity reaction to abacavir. The objectives of this study were to evaluate HLA-B*5701 prevalence among European, HIV-1-infected patients and to compare the local and central laboratory screening results. METHODS: Data were combined from six multicentre, prospective studies involving 10 European countries in which HIV-1-infected patients (irrespective of treatment experience or previous HLA-B*5701 screening), >or=18 years of age, were evaluated for HLA-B*5701 carriage, determined by the central and local laboratory methods. RESULTS: A total of 9720 patients from 272 centres were included in the analysis. The overall estimate of HLA-B*5701 prevalence in Europe was 4.98%, with country-specific estimates ranging from 1.53 to 7.75%. HLA-B*5701 prevalence was highest in the self-reported white population (6.49%) and lowest in the black population (0.39%). Local laboratory results had a high specificity (99.9%) and sensitivity (99.2%) when compared with the central laboratory results. CONCLUSION: This study supports data from previous studies regarding the prevalence of HLA-B*5701 in the HIV population and the variation of HLA-B*5701 prevalence between different racial groups. The high specificity and sensitivity of local laboratory results, suggests that clinicians can be confident in using local laboratories for pretreatment HLA-B*5701 screening. However, it is essential that local laboratories participate in HLA-B*5701-specific quality assurance programs to maintain 100% sensitivity. In HIV-infected patients, pretreatment HLA-B*5701 screening may allow more informed decisions regarding abacavir use and has the potential to significantly reduce the frequency of abacavir-related hypersensitivity reactions and costs associated with managing these reactions.

Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8(+) T cells with detection by ELISPOT and HLA-multimer staining.

Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R (2) = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.

HLA class I - associated immunodominance affects CTL responsiveness to an ESO recombinant protein tumor antigen vaccine.

PURPOSE: Vaccination with full-length human tumor antigens aims at inducing or increasing antitumor immune responses, including CD8 CTL in cancer patients across the HLA barrier. We have recently reported that vaccination with a recombinant tumor-specific NY-ESO-1 (ESO) protein, administered with Montanide and CpG resulted in the induction of specific integrated antibody and CD4 T cell responses in all vaccinated patients examined, and significant CTL responses in half of them. Vaccine-induced CTL mostly recognized a single immunodominant region (ESO 81-110). The purpose of the present study was to identify genetic factor(s) distinguishing CTL responders from nonresponders. EXPERIMENTAL DESIGN: We determined the HLA class I alleles expressed by CTL responders and nonresponders using high-resolution molecular typing. Using short overlapping peptides spanning the ESO immunodominant CTL region and HLA class I/ESO peptide tetramers, we determined the epitopes recognized by the majority of vaccine-induced CTL. RESULTS: CTL induced by vaccination with ESO protein mostly recognized distinct but closely overlapping epitopes restricted by a few frequently expressed HLA-B35 and HLA-Cw3 alleles. All CTL responders expressed at least one of the identified alleles, whereas none of the nonresponders expressed them. CONCLUSIONS: Expression of HLA-B35 and HLA-Cw3 is associated with the induction of immunodominant CTL responses following vaccination with recombinant ESO protein. Because recombinant tumor-specific proteins are presently among the most promising candidate anticancer vaccines, our findings indicate that the monitoring of cancer vaccine trials should systematically include the assessment of HLA association with responsiveness.

Crystal structures of two closely related but antigenically distinct HLA-A2/melanocyte-melanoma tumor-antigen peptide complexes.

We have determined high-resolution crystal structures of the complexes of HLA-A2 molecules with two modified immunodominant peptides from the melanoma tumor-associated protein Melan-A/Melanoma Ag recognized by T cells-1. The two peptides, a decamer and nonamer with overlapping sequences (ELAGIGILTV and ALGIGILTV), are modified in the second residue to increase their affinity for HLA-A2. The modified decamer is more immunogenic than the natural peptide and a candidate for peptide-based melanoma immunotherapy. The crystal structures at 1.8 and 2.15 A resolution define the differences in binding modes of the modified peptides, including different clusters of water molecules that appear to stabilize the peptide-HLA interaction. The structures suggest both how the wild-type peptides would bind and how three categories of cytotoxic T lymphocytes with differing fine specificity might recognize the two peptides.

Anti-HLA antibody repertoire after IVIg infusion in highly sensitized patients waiting for kidney transplantation.

Polyclonal intravenous immunoglobulin (IVIg) treatment reduces crossmatch positivity and increases rates of transplantation in highly sensitised patients (HS). We quantified the panel reactive antibody (PRA) by microlymphocytotoxicity (MLCC), and we analysed anti-HLA class I and class II IgG specific antibody repertoire by Luminex before and after IVIg infusion alone in HS patients awaiting kidney transplantation. Five patients received three monthly infusions of 1 g/kg of IVIg. Serum samples collected pre and post IVIg treatment were submitted for PRA analysis by MLCC. Anti-class I and anti-class II antibody specificities were then tested by Luminex. We focused on the anti-HLA class I and class II antibodies directed against HLA expressed by a previous graft. We also analysed the anti-HLA antibody repertoire in three patients who had not received IVIg infusion. The PRA level determined by MLCC decreased significantly in one of the five patients, dropping from 40% to 17%. The Luminex assay showed fluctuations of the anti-HLA antibody levels over time, but no significant longterm modifications of the anti-HLA antibody repertoire were observed, even in the patient with a strong and prolonged reduction of the PRA determined by MLCC. Our results show that IVIg at 1 g/kg is not sufficient to reduce PRA and does not modify the repertoire of specific anti-HLA antibody determined by Luminex.

HLA-Bw4 homozygosity is associated with an impaired CD4 T cell recovery after initiation of antiretroviral therapy.

We assessed the influence of human leukocyte antigen (HLA) alleles HLA-Bw4 and HLA-Bw6 on CD4 T cell recovery after starting successful combination antiretroviral therapy in 265 individuals. The median gains in the CD4 T cell count after 4 years were 258 cells/microL for HLA-Bw4 homozygotes, 321 cells/microL for HLA-Bw4/Bw6 heterozygotes, and 363 cells/microL for HLA-Bw6 homozygotes (P = .01, compared with HLA-Bw4 homozygotes). HLA-Bw4 homozygosity appears to predict an impaired CD4 T cell recovery after initiation of combination antiretroviral therapy.

Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide.

Immunogenicity of a long 20-mer NY-ESO-1f peptide vaccine was evaluated in a lung cancer patient TK-f01, immunized with the peptide with Picibanil OK-432 and Montanide ISA-51. We showed that internalization of the peptide was necessary to present CD8 T-cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T-cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01-restricted CD8 T-cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02-restricted CD8 T-cell clones were defined and peptide/HLA tetramers were produced. NY-ESO-1 91-101 on A*24:02, NY-ESO-1 92-102 on B*35:01, NY-ESO-1 96-104 on B*52:01 and NY-ESO-1 96-104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T-cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20-mer NY-ESO-1f peptide harboring multiple CD8 T-cell epitopes as an NY-ESO-1 vaccine. Characterization of CD8 T-cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T-cell responses comprised the dominant response.