The Length Distribution of Class I-Restricted T Cell Epitopes Is Determined by Both Peptide Supply and MHC Allele-Specific Binding Preference

HLA class I-binding predictions are widely used to identify candidate peptide targets of human CD8+ T cell responses. Many such approaches focus exclusively on a limited range of peptide lengths, typically 9 aa and sometimes 9-10 aa, despite multiple examples of dominant epitopes of other lengths. In this study, we examined whether epitope predictions can be improved by incorporating the natural length distribution of HLA class I ligands. We found that, although different HLA alleles have diverse length-binding preferences, the length profiles of ligands that are naturally presented by these alleles are much more homogeneous. We hypothesized that this is due to a defined length profile of peptides available for HLA binding in the endoplasmic reticulum. Based on this, we created a model of HLA allele-specific ligand length profiles and demonstrate how this model, in combination with HLA-binding predictions, greatly improves comprehensive identification of CD8+ T cell epitopes.Fil: Trolle, Thomas. Technical University of Denmark; DinamarcaFil: McMurtrey, Curtis. Oklahoma State University; Estados UnidosFil: Sidney, John. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Bardet, Wilfried. Oklahoma State University; Estados UnidosFil: Osborn, Sean C.. Oklahoma State University; Estados UnidosFil: Kaever, Thomas. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Sette, Alessandro. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Hildebrand, Willliam H.. Oklahoma State University; Estados UnidosFil: Nielsen, Morten. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina. Universidad Nacional de San Martín; ArgentinaFil: Peters, Bjoern. La Jolla Institute for Allergy and Immunology; Estados Unido

HLA class I molecules consistently present internal influenza epitopes

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*0702-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 (NP418-426 and NP 473-481) and from the internal viral polymerase subunit PB1 (PB1 329-337) were presented by B*0702 following infection with each of 3 different influenza strains; ligands NP418-426, NP 473-481, and PB1329-337 derived from internal viral proteins were consistently revealed by class I HLA. In contrast, ligands derived from hemagglutinin (HA) and matrix protein (M1) were presented intermittently on a strain-by-strain basis. When tested for immune recognition, HLA-B*0702 transgenic mice responded to NP418-426 and PB1 329-337 consistently and NP473-481 intermittently while ligands from HA and M1 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

<i>Toxoplasma gondii</i> peptide ligands open the gate of the HLA class I binding groove

HLA class I presentation of pathogen-derived peptide ligands is essential for CD8+ T-cell recognition of Toxoplasma gondii infected cells. Currently, little data exist pertaining to peptides that are presented after T. gondii infection. Herein we purify HLA-A*02:01 complexes from T. gondii infected cells and characterize the peptide ligands using LCMS. We identify 195 T. gondii encoded ligands originating from both secreted and cytoplasmic proteins. Surprisingly, T. gondii ligands are significantly longer than uninfected host ligands, and these longer pathogen-derived peptides maintain a canonical N-terminal binding core yet exhibit a C-terminal extension of 1-30 amino acids. Structural analysis demonstrates that binding of extended peptides opens the HLA class I F' pocket, allowing the C-terminal extension to protrude through one end of the binding groove. In summary, we demonstrate that unrealized structural flexibility makes MHC class I receptive to parasite-derived ligands that exhibit unique C-terminal peptide extensions

Identification of Class I HLA T Cell Control Epitopes for West Nile Virus

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. © 2013 Kaabinejadian et al

Determination of Cellular Lipids Bound to Human CD1d Molecules

CD1 molecules are glycoproteins that present lipid antigens at the cell surface for immunological recognition by specialized populations of T lymphocytes. Prior experimental data suggest a wide variety of lipid species can bind to CD1 molecules, but little is known about the characteristics of cellular ligands that are selected for presentation. Here we have molecularly characterized lipids bound to the human CD1d isoform. Ligands were eluted from secreted CD1d molecules and separated by normal phase HPLC, then characterized by mass spectroscopy. A total of 177 lipid species were molecularly identified, comprising glycerophospholipids and sphingolipids. The glycerophospholipids included common diacylglycerol species, reduced forms known as plasmalogens, lyso-phospholipids (monoacyl species), and cardiolipins (tetraacyl species). The sphingolipids included sphingomyelins and glycosylated forms, such as the ganglioside GM3. These results demonstrate that human CD1d molecules bind a surprising diversity of lipid structures within the secretory pathway, including compounds that have been reported to play roles in cancer, autoimmune diseases, lipid signaling, and cell death

T-Cell Tolerance for Variability in an HLA Class I-Presented Influenza A Virus Epitope▿

To escape immune recognition, viruses acquire amino acid substitutions in class I human leukocyte antigen (HLA)-presented cytotoxic T-lymphocyte (CTL) epitopes. Such viral escape mutations may (i) prevent peptide processing, (ii) diminish class I HLA binding, or (iii) alter T-cell recognition. Because residues 418 to 426 of the hypervariable influenza A virus nucleoprotein (NP418-426) epitope are consistently bound by class I HLA and presented to CTL, we assessed the impact that intraepitope sequence variability has upon T-cell recognition. CTL elicited by intranasal influenza virus infection were tested for their cross-recognition of 20 natural NP418-426 epitope variants. Six of the variant epitopes, of both H1N1 and H3N2 origin, were cross-recognized by CTL while the remaining NP418-426 epitope variants escaped targeting. A pattern emerged whereby variability at position 5 (P5) within the epitope reduced T-cell recognition, changes at P4 or P6 enabled CTL escape, and a mutation at P8 enhanced T-cell recognition. These data demonstrate that substitutions at P4 and/or P6 facilitate influenza virus escape from T-cell recognition and provide a model for the number, nature, and location of viral mutations that influence T-cell cross-recognition

Rhesus Macaque MHC Class I Molecules Present HLA-B-Like Peptides

SIV-infected Indian rhesus macaques (Macaca mulatta) are an important animal model for humans infected with HIV. Understanding macaque (M. mulatta class I (Mamu)) MHC class I-peptide binding facilitates the comparison of SIV- and HIV-specific cellular immune responses. In this study, we characterized the endogenous peptide-binding properties of three Mamu-A (A*02, A*08, A*11) and three Mamu-B (B*01, B*03, B*12) class I molecules. Motif comparisons revealed that five of the six macaque class I molecules (A*02, A*08, A*11, B*01, and B*03) have peptide-binding motifs similar to those of human class I molecules. Of the 65 macaque endogenous peptide ligands that we sequenced by tandem mass spectroscopy, 5 were previously eluted from HLA class I molecules. Nonamers predominated among the individual ligands, and both the motifs and the individual ligands indicated P2, P9, and various ancillary anchors. Interestingly, peptide binding of the Mamu-A and Mamu-B molecules exhibited cross-species peptide-presentation overlap primarily with HLA-B molecules. Indeed, all of the macaque class I molecules appeared HLA-B-like in peptide presentation. Remarkably, the overlap in macaque- and HLA-peptide presentation occurred despite divergent class I peptide-binding grooves. Macaque and human class I differing by up to 42 aa (13-23%) within the alpha-1 and alpha-2 domains, including substantial divergence within specificity pockets A-F, bound the same endogenous peptide. Therefore, endogenous peptide characterization indicates that macaque class I molecules may be the functional equivalents of HLA-B molecules

NetH2pan: A computational tool to guide MHC peptide prediction on murine tumors

With the advancement of personalized cancer immunotherapies, new tools are needed to identify tumor antigens and evaluate T-cell responses in model systems, specifically those that exhibit clinically relevant tumor progression. Key transgenic mouse models of breast cancer are generated and maintained on the FVB genetic background, and one such model is the mouse mammary tumor virus-polyomavirus middle T antigen (MMTV-PyMT) mouse-an immunocompetent transgenic mouse that exhibits spontaneous mammary tumor development and metastasis with high penetrance. Backcrossing the MMTV-PyMT mouse from the FVB strain onto a C57BL/6 genetic background, in order to leverage well-developed C57BL/6 immunologic tools, results in delayed tumor development and variable metastatic phenotypes.Therefore, we initiated characterization of the FVB MHC class I H-2q haplotype to establish useful immunologic tools for evaluating antigen specificity in the murine FVB strain. Our study provides the first detailed molecular and immunoproteomic characterization of the FVB H-2q MHC class I alleles, including >8,500 unique peptide ligands, a multiallele murine MHC peptide prediction tool, and in vivo validation of these data using MMTV-PyMT primary tumors. This work allows researchers to rapidly predict H-2 peptide ligands for immune testing, including, but not limited to, the MMTV-PyMT model for metastatic breast cancer.Fil: DeVette, Christa I.. Oklahoma Medical Research Foundation; Estados UnidosFil: Andreatta, Massimo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Bardet, Wilfried. Oklahoma Medical Research Foundation; Estados UnidosFil: Cate, Steven J.. Oklahoma Medical Research Foundation; Estados UnidosFil: Jurtz, Vanessa I.. Technical University of Denmark; DinamarcaFil: Jackson, Kenneth W.. Oklahoma Medical Research Foundation; Estados UnidosFil: Welm, Alana L.. University of Utah; Estados UnidosFil: Nielsen, Morten. Technical University of Denmark; DinamarcaFil: Hildebrand, William H.. Universidad Nacional de San Martín; Argentin