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1.
Cancer Immunol Res ; 6(6): 636-644, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29615400

RESUMO

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. Cancer Immunol Res; 6(6); 636-44. ©2018 AACR.


Assuntos
Biologia Computacional/métodos , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Antígenos de Histocompatibilidade/imunologia , Neoplasias/imunologia , Peptídeos/imunologia , Software , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cromatografia Líquida , Modelos Animais de Doenças , Feminino , Antígenos H-2/química , Antígenos H-2/genética , Antígenos H-2/imunologia , Haplótipos , Humanos , Ligantes , Neoplasias Mamárias Animais , Neoplasias Mamárias Experimentais , Espectrometria de Massas , Camundongos , Ligação Proteica
2.
PLoS One ; 12(11): e0188288, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29176828

RESUMO

HLA-E is a non-conventional MHC Class I molecule that has been recently demonstrated to present pathogen-derived ligands, resulting in the TCR-dependent activation of αß CD8+ T cells. The goal of this study was to characterize the ligandome displayed by HLA-E following infection with Mycobacterium tuberculosis (Mtb) using an in-depth mass spectrometry approach. Here we identified 28 Mtb ligands derived from 13 different source proteins, including the Esx family of proteins. When tested for activity with CD8+ T cells isolated from sixteen donors, nine of the ligands elicited an IFN-γ response from at least one donor, with fourteen of 16 donors responding to the Rv0634A19-29 peptide. Further evaluation of this immunodominant peptide response confirmed HLA-E restriction and the presence of Rv0634A19-29-reactive CD8+ T cells in the peripheral blood of human donors. The identification of an Mtb HLA-E ligand that is commonly recognized may provide a target for a non-traditional vaccine strategy.


Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Mycobacterium tuberculosis/imunologia , Peptídeos/imunologia , Tuberculose/imunologia , Tuberculose/microbiologia , Células A549 , Adulto , Sequência de Aminoácidos , Humanos , Ligantes , Peptídeos/química , Solubilidade , Especificidade da Espécie , Antígenos HLA-E
3.
J Immunol ; 196(10): 4263-73, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-27183642

RESUMO

Class I HLA molecules mark infected cells for immune targeting by presenting pathogen-encoded peptides on the cell surface. Characterization of viral peptides unique to infected cells is important for understanding CD8(+) T cell responses and for the development of T cell-based immunotherapies. Having previously reported a series of West Nile virus (WNV) epitopes that are naturally presented by HLA-A*02:01, in this study we generated TCR mimic (TCRm) mAbs to three of these peptide/HLA complexes-the immunodominant SVG9 (E protein), the subdominant SLF9 (NS4B protein), and the immunorecessive YTM9 (NS3 protein)-and used these TCRm mAbs to stain WNV-infected cell lines and primary APCs. TCRm staining of WNV-infected cells demonstrated that the immunorecessive YTM9 appeared several hours earlier and at 5- to 10-fold greater density than the more immunogenic SLF9 and SVG9 ligands, respectively. Moreover, staining following inhibition of the TAP demonstrated that all three viral ligands were presented in a TAP-dependent manner despite originating from different cellular compartments. To our knowledge, this study represents the first use of TCRm mAbs to define the kinetics and magnitude of HLA presentation for a series of epitopes encoded by one virus, and the results depict a pattern whereby individual epitopes differ considerably in abundance and availability. The observations that immunodominant ligands can be found at lower levels and at later time points after infection suggest that a reevaluation of the factors that combine to shape T cell reactivity may be warranted.


Assuntos
Apresentação de Antígeno , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Epitopos Imunodominantes/imunologia , Vírus do Nilo Ocidental/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Linhagem Celular Tumoral , Células Dendríticas/virologia , Feminino , Antígenos HLA-A/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Antígenos de Linfócitos T/imunologia
4.
Elife ; 52016 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-26824387

RESUMO

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.


Assuntos
Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Monócitos/imunologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Toxoplasma/química , Toxoplasma/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/metabolismo , Linhagem Celular , Humanos , Modelos Moleculares , Monócitos/parasitologia , Ligação Proteica , Conformação Proteica
5.
J Immunol ; 196(4): 1480-7, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26783342

RESUMO

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.


Assuntos
Mapeamento de Epitopos/métodos , Epitopos de Linfócito T/análise , Genes MHC Classe I , Antígenos HLA-A/imunologia , Antígenos HLA-B/imunologia , Peptídeos/imunologia , Alelos , Animais , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígenos HLA-A/genética , Antígenos HLA-A/metabolismo , Antígenos HLA-B/genética , Antígenos HLA-B/metabolismo , Células HeLa , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Ligantes , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica
6.
Mol Cancer Ther ; 15(2): 313-22, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26719579

RESUMO

T cells recognize cancer cells via HLA/peptide complexes, and when disease overtakes these immune mechanisms, immunotherapy can exogenously target these same HLA/peptide surface markers. We previously identified an HLA-A2-presented peptide derived from macrophage migration inhibitory factor (MIF) and generated antibody RL21A against this HLA-A2/MIF complex. The objective of the current study was to assess the potential for targeting the HLA-A2/MIF complex in ovarian cancer. First, MIF peptide FLSELTQQL was eluted from the HLA-A2 of the human cancerous ovarian cell lines SKOV3, A2780, OV90, and FHIOSE118hi and detected by mass spectrometry. By flow cytometry, RL21A was shown to specifically stain these four cell lines in the context of HLA-A2. Next, partially matched HLA-A*02:01+ ovarian cancer (n = 27) and normal fallopian tube (n = 24) tissues were stained with RL21A by immunohistochemistry to assess differential HLA-A2/MIF complex expression. Ovarian tumor tissues revealed significantly increased RL21A staining compared with normal fallopian tube epithelium (P < 0.0001), with minimal staining of normal stroma and blood vessels (P < 0.0001 and P < 0.001 compared with tumor cells) suggesting a therapeutic window. We then demonstrated the anticancer activity of toxin-bound RL21A via the dose-dependent killing of ovarian cancer cells. In summary, MIF-derived peptide FLSELTQQL is HLA-A2-presented and recognized by RL21A on ovarian cancer cell lines and patient tumor tissues, and targeting of this HLA-A2/MIF complex with toxin-bound RL21A can induce ovarian cancer cell death. These results suggest that the HLA-A2/MIF complex should be further explored as a cell-surface target for ovarian cancer immunotherapy.


Assuntos
Biomarcadores Tumorais/metabolismo , Antígeno HLA-A2/metabolismo , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Neoplasias Ovarianas/metabolismo , Peptídeos/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Afinidade de Anticorpos , Especificidade de Anticorpos , Linhagem Celular Tumoral , Feminino , Antígeno HLA-A2/imunologia , Humanos , Neoplasias Ovarianas/patologia
7.
J Virol ; 88(22): 12992-3004, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25165114

RESUMO

UNLABELLED: Identification of CD8(+) cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4(+) SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)-mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4(+) T cells into strategies designed to enhance T cell immunity. IMPORTANCE: HIV-1 epitopes catalogued by the Los Alamos National Laboratory (LANL) have yielded limited success in vaccine trials. Because the HLA of infected cells have not previously been assessed for HIV-1 ligands, the objective here was to directly characterize the viral ligands that mark infected cells. Recovery of HLA-presented peptides from HIV-1-infected CD4(+) T cells and interrogation of the peptide cargo by mass spectrometric DLS show that typical and atypical viral ligands are efficiently presented by HLA and targeted by human CTLs. Nef and Gag ligands dominate the infected cell's antigenic profile, largely due to extensive ligand sampling from select hot spots within these viral proteins. Also, HIV-1 ligands are often longer than expected, and these length variants are quite antigenic. These findings emphasize that an HLA-based view of HIV-1 ligand presentation to CTLs provides previously unrealized information that may enhance the development of immune therapies and vaccines.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Epitopos/análise , HIV-1/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/análise , Proteínas Virais/análise , Cromatografia Líquida , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Espectrometria de Massas , Peptídeos/imunologia , Proteínas Virais/imunologia
8.
Hum Immunol ; 75(3): 261-70, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24269696

RESUMO

Immunizing events including pregnancy, transfusions, and transplantation promote strong alloantibody responses to HLA. Such alloantibodies to HLA preclude organ transplantation, foster hyperacute rejection, and contribute to chronic transplant failure. Diagnostic antibody-screening assays detect alloreactive antibodies, yet key attributes including antibody concentration and isotype remain largely unexplored. The goal here was to provide a detailed profile of allogeneic antibodies to class II HLA. Methodologically, alloantibodies were purified from sensitized patient sera using an HLA-DR11 immunoaffinity column and subsequently categorized. Antibodies to DR11 were found to fix complement, exist at a median serum concentration of 2.3µg/mL, consist of all isotypes, and isotypes IgG2, IgM, and IgE were elevated. Because multimeric isotypes can confound diagnostic determinations of antibody concentration, IgM and IgA isotypes were removed and DR11-IgG tested alone. Despite removal of multimeric isotypes, patient-to-patient antibody concentrations did not correlate with MFI values. In conclusion, allogeneic antibody responses to DR11 are comprised of all antibody isotypes at differing proportions, these combined isotypes fix complement at nominal serum concentrations, and enhancements other than the removal of IgM and IgA multimeric isotypes may be required if MFI is to be used as a means of determining anti-HLA serum antibody concentrations in diagnostic clinical assays.


Assuntos
Rejeição de Enxerto/imunologia , Cadeias HLA-DRB1/metabolismo , Isotipos de Imunoglobulinas/isolamento & purificação , Imunoglobulinas/isolamento & purificação , Isoantígenos/metabolismo , Transplante de Rim , Adulto , Idoso , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular , Cromatografia de Afinidade , Proteínas do Sistema Complemento/metabolismo , Feminino , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/etiologia , Cadeias HLA-DRB1/genética , Cadeias HLA-DRB1/imunologia , Humanos , Isotipos de Imunoglobulinas/sangue , Isotipos de Imunoglobulinas/imunologia , Imunoglobulinas/sangue , Imunoglobulinas/imunologia , Testes Imunológicos , Isoantígenos/genética , Isoantígenos/imunologia , Masculino , Pessoa de Meia-Idade , Transgenes/genética , Transplante , Adulto Jovem
9.
PLoS One ; 8(6): e66298, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23762485

RESUMO

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.


Assuntos
Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Fragmentos de Peptídeos/imunologia , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/imunologia , Adulto , Idoso , Western Blotting , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Febre do Nilo Ocidental/prevenção & controle , Febre do Nilo Ocidental/virologia , Adulto Jovem
10.
Immunogenetics ; 63(12): 789-807, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21732180

RESUMO

Virus-specific CD8(+) T cells play an important role in controlling HIV/SIV replication. These T cells recognize intracellular pathogen-derived peptides displayed on the cell surface by individual MHC class I molecules. In the SIV-infected rhesus macaque model, five Mamu class I alleles have been thoroughly characterized with regard to peptide binding, and a sixth was shown to be uninvolved. In this study, we describe the peptide binding of Mamu-A1*007:01 (formerly Mamu-A*07), an allele present in roughly 5.08% of Indian-origin rhesus macaques (n = 63 of 1,240). We determined a preliminary binding motif by eluting and sequencing endogenously bound ligands. Subsequently, we used a positional scanning combinatorial library and panels of single amino acid substitution analogs to further characterize peptide binding of this allele and derive a quantitative motif. Using this motif, we selected and tested 200 peptides derived from SIV(mac)239 for their capacity to bind Mamu-A1*007:01; 33 were found to bind with an affinity of 500 nM or better. We then used PBMC from SIV-infected or vaccinated but uninfected, A1*007:01-positive rhesus macaques in IFN-γ Elispot assays to screen the peptides for T-cell reactivity. In all, 11 of the peptides elicited IFN-γ(+) T-cell responses. Six represent novel A1*007:01-restricted epitopes. Furthermore, both Sanger and ultradeep pyrosequencing demonstrated the accumulation of amino acid substitutions within four of these six regions, suggestive of selective pressure on the virus by antigen-specific CD8(+) T cells. Thus, it appears that Mamu-A1*007:01 presents SIV-derived peptides to antigen-specific CD8(+) T cells and is part of the immune response to SIV(mac)239.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T , Genes MHC Classe I/genética , Antígenos de Histocompatibilidade Classe I/genética , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Alelos , Sequência de Aminoácidos , Animais , Epitopos de Linfócito T/análise , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/análise , Antígenos de Histocompatibilidade Classe I/imunologia , Interferon gama , Macaca mulatta , Ligação Proteica , RNA Viral/sangue , RNA Viral/genética , Análise de Sequência de Proteína , Vírus da Imunodeficiência Símia/classificação , Vírus da Imunodeficiência Símia/patogenicidade , Linfócitos T Citotóxicos/imunologia , Carga Viral , Vacinas Virais
11.
Hum Immunol ; 71(1): 14-22, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19748539

RESUMO

Class I HLA sample and display peptides from thousands of endogenous proteins at the cell surface. During infection, the influenza virus modifies the host cell proteome by triggering host antiviral responses, hijacking host processes, and inhibiting host mRNA processing. In turn, the catalog of HLA class I peptides that decorate the surface of an infected cell is positioned to reflect an altered host cell proteome. To understand the host-encoded peptides presented by class I molecules after influenza infection, we compared by mass spectrometry (MS) the peptides eluted from the HLA of naive and infected cells. We identified 20 peptide ligands unique to infected cells and 347 peptides with increased presentation after infection. Infection with different influenza strains demonstrated that proteome changes are predominantly strain-specific, with few individual cellular interactions observed for multiple viral strains. Modeling by pathway analysis, however, revealed that strain specific host peptide changes represent different routes to the same destination; host changes mediated by influenza are found predominantly clustered around HLA-B, ACTB, HSP90AB1, CDK2, and ANXA2. The class I HLA proteome scanning of influenza-infected cells therefore indicates how divergent strains of influenza pursue alternate routes to access the same host cell processes.


Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Vírus da Influenza A/fisiologia , Influenza Humana/imunologia , Proteoma/análise , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Linhagem Celular , Cães , Células HeLa , Antígenos de Histocompatibilidade Classe I/química , Humanos , Ligantes , Especificidade da Espécie
12.
J Immunol ; 182(12): 7763-75, 2009 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-19494300

RESUMO

HLA-B27- and -B57-positive HIV-infected humans have long been associated with control of HIV replication, implying that CD8(+) T cell responses contribute to control of viral replication. In a similar fashion, 50% of Mamu-B*08-positive Indian rhesus macaques control SIVmac239 replication and become elite controllers with chronic-phase viremia <1000 viral RNA copies/ml. Interestingly, Mamu-B*08-restricted SIV-derived epitopes appeared to match the peptide binding profile for HLA-B*2705 in humans. We therefore defined a detailed peptide-binding motif for Mamu-B*08 and investigated binding similarities between the macaque and human MHC class I molecules. Analysis of a panel of approximately 900 peptides revealed that despite substantial sequence differences between Mamu-B*08 and HLA-B*2705, the peptide-binding repertoires of these two MHC class I molecules share a remarkable degree of overlap. Detailed knowledge of the Mamu-B*08 peptide-binding motif enabled us to identify six additional novel Mamu-B*08-restricted SIV-specific CD8(+) T cell immune responses directed against epitopes in Gag, Vpr, and Env. All 13 Mamu-B*08-restricted epitopes contain an R at the position 2 primary anchor and 10 also possess either R or K at the N terminus. Such dibasic peptides are less prone to cellular degradation. This work highlights the relevance of the Mamu-B*08-positive SIV-infected Indian rhesus macaque as a model to examine elite control of immunodeficiency virus replication. The remarkable similarity of the peptide-binding motifs and repertoires for Mamu-B*08 and HLA-B*2705 suggests that the nature of the peptide bound by the MHC class I molecule may play an important role in control of immunodeficiency virus replication.


Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/imunologia , Peptídeos/metabolismo , Vírus da Imunodeficiência Símia/imunologia , Replicação Viral , Sequência de Aminoácidos , Animais , DNA Polimerase Dirigida por DNA , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/química , Humanos , Ligantes , Macaca mulatta , Dados de Sequência Molecular , Biblioteca de Peptídeos , Ligação Proteica , Homologia de Sequência de Aminoácidos
13.
J Virol ; 83(18): 9206-14, 2009 09.
Artigo em Inglês | MEDLINE | ID: mdl-19553306

RESUMO

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 (NP(418-426)) 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 NP(418-426) epitope variants. Six of the variant epitopes, of both H1N1 and H3N2 origin, were cross-recognized by CTL while the remaining NP(418-426) 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.


Assuntos
Epitopos/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Tolerância Imunológica , Vírus da Influenza A/imunologia , Mutação de Sentido Incorreto , Proteínas de Ligação a RNA/imunologia , Linfócitos T/imunologia , Proteínas do Core Viral/imunologia , Apresentação de Antígeno , Antígenos Virais , Apresentação Cruzada , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A Subtipo H3N2 , Proteínas do Nucleocapsídeo
14.
PLoS One ; 4(5): e5325, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19415116

RESUMO

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.


Assuntos
Antígenos CD1d/metabolismo , Lipídeos/análise , Cromatografia Líquida de Alta Pressão , Glicerofosfolipídeos/análise , Humanos , Espectrometria de Massas , Ligação Proteica , Esfingolipídeos/análise
15.
J Virol ; 83(12): 6011-9, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19339351

RESUMO

Vaccines that elicit CD8(+) T-cell responses are routinely tested for immunogenicity in nonhuman primates before advancement to clinical trials. Unfortunately, the magnitude and specificity of vaccine-elicited T-cell responses are variable in currently utilized nonhuman primate populations, owing to heterogeneity in major histocompatibility (MHC) class I genetics. We recently showed that Mauritian cynomolgus macaques (MCM) have unusually simple MHC genetics, with three common haplotypes encoding a shared pair of MHC class IA alleles, Mafa-A*25 and Mafa-A*29. Based on haplotype frequency, we hypothesized that CD8(+) T-cell responses restricted by these MHC class I alleles would be detected in nearly all MCM. We examine here the frequency and functionality of these two alleles, showing that 88% of MCM express Mafa-A*25 and Mafa-A*29 and that animals carrying these alleles mount three newly defined simian immunodeficiency virus-specific CD8(+) T-cell responses. The epitopes recognized by each of these responses accumulated substitutions consistent with immunologic escape, suggesting these responses exert antiviral selective pressure. The demonstration that Mafa-A*25 and Mafa-A*29 restrict CD8(+) T-cell responses that are shared among nearly all MCM indicates that these animals are an advantageous nonhuman primate model for comparing the immunogenicity of vaccines that elicit CD8(+) T-cell responses.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Macaca fascicularis/genética , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Alelos , Substituição de Aminoácidos , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/virologia , Epitopos de Linfócito T/imunologia , Frequência do Gene , Haplótipos , Antígenos de Histocompatibilidade Classe I/imunologia , Macaca fascicularis/imunologia , Repetições de Microssatélites , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia
16.
Proc Natl Acad Sci U S A ; 106(2): 540-5, 2009 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-19122146

RESUMO

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 (NP(418-426) 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 NP(418-426), NP(473-481), and PB1(329-337) 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 NP(418-426) and PB1(329-337) consistently and NP(473-481) 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.


Assuntos
Apresentação de Antígeno/imunologia , Antígenos Virais/imunologia , Antígenos HLA-B/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Orthomyxoviridae/imunologia , Animais , Antígenos Virais/análise , Antígenos HLA/imunologia , Antígeno HLA-B7 , Células HeLa , Humanos , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Nucleoproteínas/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas do Core Viral/imunologia , Proteínas Virais/imunologia
17.
J Proteome Res ; 7(4): 1445-57, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18345606

RESUMO

Cellular immune mechanisms detect and destroy cancerous and infected cells via the human leukocyte antigen (HLA) class I molecules that present peptides of intracellular origin on the surface of all nucleated cells. The identification of novel, tumor-specific epitopes is a critical step in the development of immunotherapeutics for breast cancer. To directly identify peptide epitopes unique to cancerous cells, secreted human class I HLA molecules (sHLA) were constructed by deletion of the transmembrane and cytoplasmic domain of HLA A*0201. The resulting sHLA-A*0201 was transferred and expressed in breast cancer cell lines MCF-7, MDA-MB-231, and BT-20 as well as in the immortal, nontumorigenic cell line MCF10A. Stable transfectants were seeded into bioreactors for production of > 25 mg of sHLA-A*0201. Peptides eluted from affinity purified sHLA were analyzed by mass spectroscopy. Comparative analysis of HLA-A*0201 peptides revealed 5 previously uncharacterized epitopes uniquely presented on breast cancer cells. These peptides were derived from intracellular proteins with either well-defined or putative roles in breast cancer development and progression: Cyclin Dependent Kinase 2 (Cdk2), Ornithine Decarboxylase (ODC1), Kinetochore Associated 2 (KNTC2 or HEC1), Macrophage Migration Inhibitory Factor (MIF), and Exosome Component 6 (EXOSC6). Cellular recognition of the MIF, KNTC2, EXOSC6, and Cdk2 peptides by circulating CD8+ cells was demonstrated by tetramer staining and IFN-gamma ELISPOT. The identification and characterization of peptides unique to the class I of breast cancer cells provide putative targets for the development of immune diagnostic tools and therapeutics.


Assuntos
Neoplasias da Mama/imunologia , Epitopos de Linfócito T/análise , Antígenos HLA-A/imunologia , Sequência de Aminoácidos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Quinase 2 Dependente de Ciclina/imunologia , Quinase 2 Dependente de Ciclina/metabolismo , Proteínas do Citoesqueleto , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/isolamento & purificação , Exorribonucleases/imunologia , Exorribonucleases/metabolismo , Feminino , Antígenos HLA-A/química , Antígeno HLA-A2 , Humanos , Interferon gama/metabolismo , Oxirredutases Intramoleculares/imunologia , Oxirredutases Intramoleculares/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Fatores Inibidores da Migração de Macrófagos/imunologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Espectrometria de Massas , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Ornitina Descarboxilase/imunologia , Ornitina Descarboxilase/metabolismo
18.
Proc Natl Acad Sci U S A ; 105(8): 2981-6, 2008 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-18299564

RESUMO

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.


Assuntos
Epitopos de Linfócito T/genética , Antígenos HLA/metabolismo , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/imunologia , Animais , Sequência de Bases , Chlorocebus aethiops , Primers do DNA/genética , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Antígenos HLA/imunologia , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência , Células Vero , Vírus do Nilo Ocidental/genética
19.
J Immunol ; 175(9): 5986-97, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16237093

RESUMO

Although the SIV-infected Indian rhesus macaque (Macaca mulatta) is the animal model most widely used for studying HIV infection, our current understanding of the functional macaque MHC class I molecules is limited. To date, SIV-derived CD8+ T lymphocyte epitopes from only three high frequency macaque MHC class I molecules have been extensively characterized. In this study, we defined the peptide-binding properties of the high frequency Indian rhesus macaque class I molecule, Mamu-B*01 ( approximately 26%). We first identified a preliminary binding motif by eluting and sequencing endogenously bound Mamu-B*01 ligands. We further characterized the peptide-binding characteristics using panels of single amino acid substitution analogs. Using this detailed motif, 507 peptides derived from SIV(mac)239 were identified and tested for their Mamu-B*01 binding capacity. Surprisingly, only 11 (2.2%) of these motif-containing peptides bound with IC50 values < or =500 nM. We assessed the immunogenicity of these peptides using freshly isolated PBMC from ten Mamu-B*01+ SIV-infected rhesus macaques in IFN-gamma ELISPOT and IFN-gamma/TNF-alpha intracellular cytokine staining assays. Lymphocytes from these SIV-infected macaques responded to none of these peptides. Furthermore, there was no sequence variation indicative of escape in the regions of the virus that encoded these peptides. Additionally, we could not confirm previous reports of SIV-derived Mamu-B*01-restricted epitopes in the Env and Gag proteins. Our results suggest that the high frequency MHC class I molecule, Mamu-B*01, is not involved in SIV-specific CD8+ T lymphocyte responses.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/fisiologia , Vírus da Imunodeficiência Símia/imunologia , Alelos , Sequência de Aminoácidos , Animais , Epitopos de Linfócito T , Genes MHC Classe I , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Interferon gama/biossíntese , Macaca mulatta , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Relação Estrutura-Atividade , Proteínas Virais/metabolismo
20.
Biochemistry ; 44(37): 12491-507, 2005 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-16156661

RESUMO

Various approaches are currently proposed to successfully develop therapies for the prevention and treatment of infectious diseases and cancer. One of the most promising approaches is the development of vaccines that elicit cytotoxic T lymphocyte (CTL) responses. Consequently, identification and exact definition of molecular parameters involved in peptide-MHC class-I interactions of putative CTL epitopes are of prime importance for the development of immunomodulating compounds. To better facilitate epitope discovery, we developed and validated a novel state-of-the-art biochemical HLA-A0201 assay, which is comprised of technologically advanced cutting edge reagents. The technique is based on competition and uses a FITC-labeled reference peptide and highly purified soluble HLA-A0201 molecules to quantitatively measure the binding capacity of nonlabeled peptide candidates. Detection by fluorescence polarization allows real-time measurement of binding ratios without separation steps. During standardization, the problem of assay parameter variation is discussed, showing the dramatic influence of HLA and reference peptide concentrations as well as the choice of the reference peptide itself on IC(50) determinations. For validation, a panel of 15 well-defined HLA-A0201 ligands from various sources covering a broad range of binding affinities was tested. Binding data were used to compare against pre-existing quantitative assay systems. The results obtained demonstrated significant correlation among assay procedures, suggesting that the application of fluorescence polarization in combination with recombinant sHLA molecules is highly advantageous for the accurate assessment of peptide binding. Furthermore, the assay also features high-throughput screening capacity, providing uniquely efficient means of identifying and evaluating immune target molecules.


Assuntos
Antígenos HLA-A/química , Sítios de Ligação , Epitopos/análise , Fluoresceína-5-Isotiocianato , HIV-1/imunologia , Antígenos HLA-A/metabolismo , Antígeno HLA-A2 , Vírus da Hepatite B/imunologia , Humanos , Peptídeos/química , Peptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência
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