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Abstract: There is currently a significant interest in the identification and validation of HLA-restricted CTL epitopes, which are thought to have important implications for the development of preventive and/or therapeutic applications in bacterial or viral infections, autoimmune diseases, and cancer. To better facilitate epitope discovery and validation, we present a cell- and radioisotope-free HLA-A⁎0201 assay system which relies upon fluorescence polarization. The assay has the advantage of allowing real-time measurements in solution without separation steps. In this report, we directed our efforts towards enhancing the sensitivity and reproducibility of the assay by conducting an in-depth analysis of parameters critical for standardization. Initial experiments demonstrated that the attachment of a fluorescence moiety at positions 5 and 8 for 9-mers and positions 5 and 6 for 10-mers, respectively, does not interfere with ligand binding to soluble HLA-A⁎0201. In addition, it was found that their binding to HLA-A⁎0201 was very effective showing high affinity binding with K d''s between 10.7 to 21.8 nM and binding capacities of up to 37%. In order to deliver maximized responses, factors such as the regulation of thermal HLA activation parameters to initiate peptide exchange as well as the specific adjustment of assay components were identified. Overall, the results obtained clearly demonstrate high accuracy, sensitivity and reproducibility of the FP-based assay approach. With the need for both increased throughput and miniaturized volumes, this fully homogenous, fluorescent-type binding assay is expected to be useful for routine analysis of peptide binding to MHC class I as well as class II molecules. [Copyright &y& Elsevier]

Abstract: Major histocompatibility complex (MHC) class I molecules bind and display peptide antigens on the cell surface. CD8+ T lymphocytes recognize peptides in association with class I proteins to initiate a cytotoxic immune response. To understand the specificity of such immune responses and to facilitate the development of therapies for disease, it is important to identify MHC-presented peptides. In this study, platelets, easily obtainable and often associated with immune-mediated disease, were selected to identify MHC class I–associated peptides. MHC-associated peptides presented on platelets of normal individuals and individuals with idiopathic thrombocytopenic purpura (ITP) were characterized. ITP is characterized by the premature immune destruction of platelets. It is associated with the production of antiplatelet autoantibodies, most often targeting platelet membrane GPIIb/IIIa or GPIb/IX. In addition to characterizing five fully and several partially sequenced peptides from platelets, the peptide GPRGA(L/I)S(L/I)(L/I) was identified from four of the five ITP patients. The anchor motif of this peptide correlates with the presence of the HLA-B7 allele. A BLAST search identified this peptide as GPIb (4–12). In conclusion, platelets from normal and ITP individuals can present peptides from general cellular proteins and platelet specific proteins, such as GPIb, to the immune system via MHC class I. [Copyright &y& Elsevier]

Neoantigens are novel peptide sequences resulting from somatic mutations in tumors that upon loading onto major histocompatibility complex (MHC) molecules allow recognition by T cells. Accurate neoantigen identification is thus critical for designing cancer vaccines and predicting response to immunotherapies. Neoantigen prioritization relies on correctly inferring whether the presenting peptide sequence can successfully induce an immune response. An important, yet underappreciated, variable in neoantigen-prediction pipelines is the mutation position within the peptide relative to its anchor positions for the patient's specific HLA alleles. While a subset of peptide positions are presented to the T-cell receptor for recognition, others are responsible for anchoring to the MHC, making these positional considerations critical for predicting T-cell responses. However, a systematic method for determining anchor locations and application of these to evaluate neoantigens has not been reported. Here, we calculated high probability anchor positions for 328 HLA alleles using a reference dataset generated from clinical and TCGA patient samples. Results showed clusters of different anchor trends among the HLA alleles analyzed. A subset of anchor results were orthogonally validated using protein crystallography structures. Analysis of 923 tumor samples showed that 7-41% of neoantigen candidates were potentially misclassified in the neoantigen selection process and can be rescued using allele-specific knowledge of anchor positions. Representative anchor trends have been experimentally validated using peptide-MHC stability assays and competition binding assays. By incorporating our anchor prediction results into neoantigen prediction pipelines, we hope to formalize and streamline the identification process for relevant clinical studies. [ABSTRACT FROM AUTHOR]

The recent West Nile virus (WNV) outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1) the number of viral ligands presented by the HLA of infected cells, and 2) the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity. [ABSTRACT FROM AUTHOR]

Cytotoxic T lymphocytes (CTL) limit influenza virus replication and prevent morbidity and mortality upon recognition of HLA class I presented epitopes on the surface of virus infected cells, yet the number and origin of the viral epitopes that decorate the infected cell are unknown. To understand the presentation of influenza virus ligands by human MHC class I molecules, HLA-B*O7O2-presented viral peptides were directly identified following influenza infection. After transfection with soluble class I molecules, peptide ligands unique to infected cells were eluted from isolated MHC molecules and identified by comparative mass spectrometry (MS). Then CTL were gathered following infection with influenza and viral peptides were tested for immune recognition. We found that the class I molecule B*0702 presents 3-6 viral ligands following infection with different strains of influenza. Peptide ligands derived from the internal viral nucleoprotein (NP[sub418-426] and NP[sub473-481]) and from the internal viral polymerase subunit PB1 (PB1[sub329-337]) were presented by B*0702 following infection with each of 3 different influenza strains; ligands NP[sub418-426], NP[sub473--481], and PB1[sub329-337] derived from internal viral proteins were consistently revealed by class I HLA. In contrast, ligands derived from hemagglutinin (HA) and matrix protein (Ml) were presented intermittently on a strain-by-strain basis. When tested for immune recognition, HLA-B*O7O2 transgenic mice responded to NP[sub418-426] and PB1[sub329-337] consistently and NP[sub473-481] intermittently while ligands from HA and Ml were not recognized. These data demonstrate an emerging pattern whereby class I HLA reveal a handful of internal viral ligands and whereby CTL recognize consistently presented influenza ligands. [ABSTRACT FROM AUTHOR]

Cytotoxic T lymphocytes (CTL) play an important role in the control and elimination of infection by West Nile virus (WNV), yet the class I human leukocyte antigen (HLA)-presented peptide epitopes that enable CTL recognition of WNV-infected cells remain uncharacterized. The goals of this work were first to discover the peptide epitopes that distinguish the class I HLA of WNV-infected cells and then to test the T cell reactivity of newly discovered WNV epitopes. To discover WNV-immune epitopes, class I HLA was harvested from WNV (NY99 strain)-infected and uninfected HeLa cells. Then peptide epitopes were eluted from affinity-purified HLA, and peptide epitopes from infected and uninfected cells were comparatively mapped by mass spectroscopy. Six virus-derived peptides from five different viral proteins (E, NS2b, NS3, NS4b, and NS5) were discovered as unique to HLA-A*0201 of infected cells, demonstrating that the peptides sampled by class I HLA are distributed widely throughout the WNV proteome. When tested with CTL from infected individuals, one dominant WNV target was apparent, two epitopes were subdominant, and three demonstrated little CTL reactivity. Finally, a sequence comparison of these epitopes with the hundreds of viral isolates shows that HLA-A*0201 presents epitopes derived from conserved regions of the virus. Detection and recovery from WNV infection are therefore functions of the ability of class I HLA molecules to reveal conserved WNV epitopes to an intact cellular immune system that subsequently recognizes infected cells. [ABSTRACT FROM AUTHOR]