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

Regulation and Autoregulation of the Promoter for the Latency-associated Nuclear Antigen of Kaposi's Sarcoma-associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 has been established as the etiological agent of Kaposi's sarcoma and certain AIDS-associated lymphomas. KSHV establishes latent infection in these tumors, invariably expressing high levels of the viral latency-associated nuclear antigen (LANA) protein. LANA is necessary and sufficient to maintain the KSHV episome. It also modulates viral and cellular transcription and has been implicated directly in oncogenesis because of its ability to bind to the p53 and pRb tumor suppressor proteins. Previously, we identified the LANA promoter (LANAp) and showed that it was positively regulated by LANA itself. Here, we present a detailed mutational analysis and define cis-acting elements and trans-acting factors for the core LANAp. We found that a downstream promoter element, TATA box, and GC box/Sp1 site at -29 are all individually required for activity. This architecture places LANAp into the small and unusual group of eukaryotic promoters that contain both the downstream promoter element and TATA element but lack a defined initiation site. Furthermore, we demonstrate that LANA regulates its own promoter via its C-terminal domain and does bind to a defined site within the core promoter

<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

Variability in German Cockroach Extract Composition Greatly Impacts T Cell Potency in Cockroach-Allergic Donors

German cockroach extract is used clinically to evaluate allergen-specific sensitization and for subcutaneous allergen-specific immunotherapy, though there are no guidelines for standardization in its manufacture. We performed an immunological evaluation of 12 different cockroach extracts prepared from different sources and their potency to induce allergen-specific T cell reactivity. PBMC from 13 cockroach allergic donors were expanded in vitro with 12 different German cockroach extracts. After culture expansion, cells were re-stimulated with the different extracts and T cell responses were assessed by FluoroSpot (IL-5, IFNγ and IL-10 production). In parallel to the extracts, single allergen peptide pools for allergens from groups 1, 2, 4, 5, and 11 were tested to determine allergen immunodominance. Furthermore, to assess allergy specificity, PBMC from 13 non-allergic donors were also tested with the most potent extract and T cell responses were compared to the allergic cohort. Dramatic variations in T cell reactivity were observed to the different cockroach extract batches. Response magnitudes varied over 3 logs within a single donor. IL-5 production in the allergic cohort was significantly higher compared to the non-allergic cohort (p=0.004). Allergen content determination by ELISA detected much lower concentrations of Bla g 5 compared to Bla g 1 and 2. Mass spectrometric analysis revealed that Bla g 5 was present in similar amounts to Bla g 1 and 2 in extracts made from whole body, whereas it was not detected in extracts made from fecal matter, suggesting that Bla g 5 is not excreted into feces. Different donors exhibit different response patterns to different extracts, potentially dependent on the donor-specific T cell allergen immunodominance pattern and the allergen content of the extract tested. These findings have dramatic implications for the selection of potent extracts used for diagnostic purposes or allergen-specific immunotherapy

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

Surface Phenotype and Functionality of WNV Specific T Cells Differ with Age and Disease Severity

West Nile virus (WNV) infection can result in severe neuroinvasive disease, particularly in persons with advanced age. As rodent models demonstrate that T cells play an important role in limiting WNV infection, and strong T cell responses to WNV have been observed in humans, we postulated that inadequate antiviral T cell immunity was involved in neurologic sequelae and the more severe outcomes associated with age. We previously reported the discovery of six HLA-A*0201 restricted WNV peptide epitopes, with the dominant T cell targets in naturally infected individuals being SVG9 (Env) and SLF9 (NS4b). Here, memory phenotype and polyfunctional CD8+ T cell responses to these dominant epitopes were assessed in 40 WNV seropositive patients displaying diverse clinical symptoms. The patients' PBMC were stained with HLA-I multimers loaded with the SVG9 and SLF9 epitopes and analyzed by multicolor flow cytometry. WNV-specific CD8+ T cells were found in peripheral blood several months post infection. The number of WNV-specific T cells in older individuals was the same, if not greater, than in younger members of the cohort. WNV-specific T cells were predominantly monofunctional for CD107a, MIP-1β, TNFα, IL-2, or IFNγ. When CD8+ T cell responses were stratified by disease severity, an increased number of terminally differentiated, memory phenotype (CD45RA+ CD27− CCR7− CD57+) T cells were detected in patients suffering from viral neuroinvasion. In conclusion, T cells of a terminally differentiated/cytolytic profile are associated with neuroinvasion and, regardless of age, monofunctional T cells persist following infection. These data provide the first indication that particular CD8+ T cell phenotypes are associated with disease outcome following WNV infection

A shared MHC supertype motif emerges by convergent evolution in macaques and mice, but is totally absent in human MHC molecules

The SIV-infected rhesus macaque (Macaca mulatta) is the most established model of AIDS disease systems, providing insight into pathogenesis and a model system for testing novel vaccines. The understanding of cellular immune responses based on the identification and study of Major Histocompatibility Complex (MHC) molecules, including their MHC:peptide-binding motif, provides valuable information to decipher outcomes of infection and vaccine efficacy. Detailed characterization of Mamu-B*039:01, a common allele expressed in Chinese rhesus macaques, revealed a unique MHC:peptide-binding preference consisting of glycine at the second position. Peptides containing a glycine at the second position were shown to be antigenic from animals positive for Mamu-B*039:01. A similar motif was previously described for the Dd mouse MHC allele, but for none of the human HLA molecules for which a motif is known. Further investigation showed that one additional macaque allele, present in Indian rhesus macaques, Mamu-B*052:01, shares this same motif. These “G2” alleles were associated with the presence of specific residues in their B pocket. This pocket structure was found in 6% of macaque sequences but none of 950 human HLA class I alleles. Evolutionary studies using the “G2” alleles points to common ancestry for the macaque sequences, while convergent evolution is suggested when murine and macaque sequences are considered. This is the first detailed characterization of the pocket residues yielding this specific motif in nonhuman primates and mice, revealing a new supertype motif not present in humans

Role of MAIT cells in pulmonary bacterial infection.

Mucosal-associated invariant T (MAIT) cells represent a population of innate T cells that is highly abundant in humans. MAIT cells recognize metabolites of the microbial vitamin B pathway that are presented by the major histocompatibility complex (MHC) class I-related protein MR1. Upon bacterial infection, activated MAIT cells produce diverse cytokines and cytotoxic effector molecules and accumulate at the site of infection, thus, MAIT cells have been shown to be protective against various bacterial infections. Here, we summarize the current knowledge of the role of MAIT cells in bacterial pulmonary infection models

3-O-(3′,3′-Dimethysuccinyl) Betulinic Acid Inhibits Maturation of the Human Immunodeficiency Virus Type 1 Gag Precursor Assembled In Vitro

3-O-(3′,3′-Dimethysuccinyl) betulinic acid (PA-457) has been shown to potently inhibit human immunodeficiency virus (HIV) replication in culture. In contrast to inhibitors that act upon the viral proteinase, PA-457 appears to block only the final maturational cleavage of p25CA-p2 to p24CA. However, attempts to replicate this effect in vitro using recombinant Gag have failed, leading to the hypothesis that activity is dependent upon the assembly state of Gag. Using a synthesis/assembly system for chimeric HIV type 1 Gag proteins, we have replicated the activity of PA-457 in vitro. The processing of assembled chimeric Gag can be inhibited by the addition of drug with only the final cleavage of p25CA-p2 to p24CA blocked. Consistent with our hypothesis and with previous findings, inhibition appears specific to Gag assembled into an immature capsid-like structure, since synthetic Gag that remains unassembled is properly processed in the presence of the compound. To further analyze the authenticity of the assay, PA-457 was tested in parallel with its inactive parental compound, betulinic acid. Betulinic acid had no effect upon p25 processing in this system. Analysis of a PA-457-resistant mutant, A1V, in this system pointed to more rapid cleavage as a possible mechanism for resistance. However, characterization of additional mutations at the cleavage site and in p2 suggests that resistance does not strictly correlate with the rate of cleavage. With the establishment of an in vitro assay for the detection of PA-457 activity, a more detailed characterization of its mechanism of action will be possible