Comparison of HLA ligand elution data and binding predictions reveals varying prediction performance for the multiple motifs recognized by HLA-DQ2.5

ABSTRACTBinding prediction tools are commonly used to identify peptides presented on MHC class II molecules. Recently, a wealth of data in the form of naturally eluted ligands has become available and discrepancies between ligand elution data and binding predictions have been reported. Quantitative metrics for such comparisons are currently lacking. In this study, we assessed how efficiently MHC class II binding predictions can identify naturally eluted peptides, and investigated instances with discrepancies between the two methods in detail. We found that, in general, MHC class II eluted ligands are predicted to bind to their reported restriction element with high affinity. But, for several studies reporting an increased number of ligands that were not predicted to bind, we found that the reported MHC restriction was ambiguous. Additional analyses determined that most of the ligands predicted to not bind are either weak binders or predicted to bind other co-expressed MHC class II molecules. For selected alleles, we addressed discrepancies between elution data and binding predictions by experimental measurements, and found that predicted and measured affinities correlate well. For DQA1*05:01/DQB1*02:01 (DQ2.5) however, binding predictions did miss several peptides that were determined experimentally to be binders. For these peptides and several known DQ2.5 binders we determined key residues for conferring DQ2.5 binding capacity, which revealed that DQ2.5 utilizes two different binding motifs, of which only one is predicted effectively. These findings have important implications for the interpretation of ligand elution data and for the improvement of MHC class II binding predictions.

A CD8+ T-Cell Clone Directed Against AML Blasts that Recognizes a Tumor Specific Antigen Expressed Also by Solid Tumors

Abstract 4289 The aim of this study was to generate cytotoxic T-lymphocytes (CTL) clones directed against AML cells and to identify new immunogenic antigens with the following properties: i) to be overexpressed by leukemic cells and not by normal tissues; ii) to be shared among different AML subtypes; iii) to play a role in leukemic growth/survival; iv) to be expressed by leukemic stem cells. To this end, we loaded normal dendritic cells (DC) from a healthy donor with apoptotic bodies from primary AML cells and used loaded DC to stimulate autologous lymphocytes (i.e. lymphocytes from the donor). Donor was selected to be partially matched with the leukemic patient for MHC-class I, in that he shared two MHC-class I alleles at the HLA-supertype level (HLA-B7 and HLA-B44). With this strategy, a CD8+ T-cell line was generated that recognized both loaded DC and AML cells used for loading, but not DC loaded with normal cells derived from the patient (PHA-blasts). This CTL line was cloned by limiting dilutions and 180 clones were screened by IFN-g elispot against 2 different AML samples that were expressing both HLA-B7 and HLA-B44; one additional mismatched AML sample was used as negative control. Two clones were selected that recognized HLA-B7+/HLA-B44+ AML cells but not the negative control (namely, clone 31D3 and 8E12). To determine the HLA-restriction element and to verify shared antigen expression among AML subtypes, we tested both clones against a panel of 18 HLA-typed primary AML samples. We found that clone 31D3 was restricted by HLA-B7 and clone 8E12 by HLA-B44. Each clone recognized 5/5 HLA-matched AML samples of different subtype. In addition, the clones did not recognize both resting and activated normal myeloid, nor lymphoid and CD34+ cells expressing the proper HLA-restriction allele. In addition to elispot activity, both clones showed killing of AML cells in a CFSE-based cytotoxic assay. To confirm the lack of reactivity against normal tissues, we analyzed the activity of both clones against HLA matched or mismatched fibroblasts and we found that clone 8E12 displayed a low reactivity activity against normal matched fibroblasts, while clone 31D3 was not reactive. We then focused the analysis on clone 31D3 and tested whether its activity was restricted to leukemia or also directed against a panel of HLA-B7 positive (N=3) or negative (N=5) solid tumors. Interestingly, we found that the clone 31D3 could recognize one colon carcinoma and one melanoma cell line expressing the HLA-B7 supertype. In particular, the melanoma cell line (G4-mel) was HLA-B35+ (HLA-B35 belongs to the HLA-B7 supertype) and was recognized at very high levels. The availability of a tumor cell line expressing adequate amounts of antigen will make the generation of a cDNA library for antigen identification more feasible than with primary leukemic cells. Finally, to further confirm that HLA-B35 is the proper MHC restriction element and to determine to what extent the antigen is shared among different tumors, we transduced 8 tumor cell lines, 3 normal B-lymphoblastoid and 1 fibroblast cell line with the HLA-B35 allele. Clone 31D3 efficiently recognized 6/8 HLA-B35 transduced tumor cell lines (1 AML, 2 melanomas, 2 colon and 1 breast carcinoma) but none of the control cell lines. In conclusion, clone 31D3 is restricted by the HLA-B7 supertype, it recognizes an antigen that is expressed by leukemic cells and not by normal tissues and, most importantly, is shared among tumors of different histology. Since the vast majority of malignant cells tested (both primary cells and in vitro established cell lines) were targeted by this clone, the putative antigen might be a protein playing an important role in tumor growth or survival. We are now testing the activity of the clone against purified leukemic stem cells and attempting to identify this shared tumor antigen. Disclosures: No relevant conflicts of interest to declare.

Characterization of the Fine Specificity of Bovine CD8 T-Cell Responses to Defined Antigens from the Protozoan Parasite Theileria parva

ABSTRACT Immunity against the bovine intracellular protozoan parasite Theileria parva has been shown to be mediated by CD8 T cells. Six antigens targeted by CD8 T cells from T. parva-immune cattle of different major histocompatibility complex (MHC) genotypes have been identified, raising the prospect of developing a subunit vaccine. To facilitate further dissection of the specificity of protective CD8 T-cell responses and to assist in the assessment of responses to vaccination, we set out to identify the epitopes recognized in these T. parva antigens and their MHC restriction elements. Nine epitopes in six T. parva antigens, together with their respective MHC restriction elements, were successfully identified. Five of the cytotoxic-T-lymphocyte epitopes were found to be restricted by products of previously described alleles, and four were restricted by four novel restriction elements. Analyses of CD8 T-cell responses to five of the epitopes in groups of cattle carrying the defined restriction elements and immunized with live parasites demonstrated that, with one exception, the epitopes were consistently recognized by animals of the respective genotypes. The analysis of responses was extended to animals immunized with multiple antigens delivered in separate vaccine constructs. Specific CD8 T-cell responses were detected in 19 of 24 immunized cattle. All responder cattle mounted responses specific for antigens for which they carried an identified restriction element. By contrast, only 8 of 19 responder cattle displayed a response to antigens for which they did not carry an identified restriction element. These data demonstrate that the identified antigens are inherently dominant in animals with the corresponding MHC genotypes.

Antigen Mining with Iterative Genome Screens Identifies Novel Diagnostics for the Mycobacterium tuberculosis Complex

ABSTRACT The definition of antigens for the diagnosis of human and bovine tuberculosis is a research priority. If diagnosis is to be used alongside Mycobacterium bovis BCG-based vaccination regimens, it will be necessary to have reagents that allow the discrimination of infected and vaccinated animals. A list of 42 potential M. bovis-specific antigens was prepared by comparative analysis of the genomes of M. bovis, M. avium subsp. avium, M. avium subsp. paratuberculosis, and Streptomyces coelicolor. Potential antigens were tested by applying them in a high-throughput peptide-based screening system to M. bovis-infected and BCG-vaccinated cattle and to cattle without prior exposure to M. bovis. A response hierarchy of antigens was established by comparing responses in infected animals. Three antigens (Mb2555, Mb2890, and Mb3895) were selected for further study, as they were strongly recognized in experimentally infected animals but with low or no frequency in BCG-vaccinated and naïve cows. Interestingly, all three antigens were recognized in animals vaccinated against Johne's disease, suggesting the presences of epitopes cross-reacting with M. avium subsp. paratuberculosis antigens. Eight peptides from the three antigens studied in detail were identified as immunodominant and were characterized in terms of major histocompatibility complex class II restriction element usage and shown to be restricted through both DR and DQ molecules. Reasons for antigenic cross-reactivity with M. avium subsp. paratuberculosis and refinement of the in silico strategy to predict such cross-reactivity from the primary protein sequence will be discussed. Evaluation of the peptides identified from the three dominant antigens by use of larger field studies is now a priority.

Components of the Ligand for a Ni++ Reactive Human T Cell Clone

The major histocompatibility complex (MHC) restriction element for a human Ni2+ reactive T cell, ANi-2.3, was identified as DR52c. A series of experiments established that the functional ligand for this T cell was a preformed complex of Ni2+ bound to the combination of DR52c and a specific peptide that was generated in human and mouse B cells, but not in fibroblasts nor other antigen processing–deficient cells. In addition, ANi-2.3 recognition of this complex was dependent on His81 of the MHC β chain, suggesting a role for this amino acid in Ni2+ binding to MHC. We propose a general model for Ni2+ recognition in which βHis81 and two amino acids from the NH2-terminal part of the MHC bound peptide coordinate Ni2+ which then interacts with some portion of the Vα CDR1 or CDR2 region.