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1.
J Immunol ; 207(12): 2913-2921, 2021 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-34810222

RESUMEN

CD8+ T cells are key mediators of antiviral and antitumor immunity. The isolation and study of Ag-specific CD8+ T cells, as well as mapping of their MHC restriction, has practical importance to the study of disease and the development of therapeutics. Unfortunately, most experimental approaches are cumbersome, owing to the highly variable and donor-specific nature of MHC-bound peptide/TCR interactions. Here we present a novel system for rapid identification and characterization of Ag-specific CD8+ T cells, particularly well suited for samples with limited primary cells. Cells are stimulated ex vivo with Ag of interest, followed by live cell sorting based on surface-trapped TNF-α. We take advantage of major advances in single-cell sequencing to generate full-length sequence data from the paired TCR α- and ß-chains from these Ag-specific cells. The paired TCR chains are cloned into retroviral vectors and used to transduce donor CD8+ T cells. These TCR transductants provide a virtually unlimited experimental reagent, which can be used for further characterization, such as minimal epitope mapping or identification of MHC restriction, without depleting primary cells. We validated this system using CMV-specific CD8+ T cells from rhesus macaques, characterizing an immunodominant Mamu-A1*002:01-restricted epitope. We further demonstrated the utility of this system by mapping a novel HLA-A*68:02-restricted HIV Gag epitope from an HIV-infected donor. Collectively, these data validate a new strategy to rapidly identify novel Ags and characterize Ag-specific CD8+ T cells, with applications ranging from the study of infectious disease to immunotherapeutics and precision medicine.


Asunto(s)
Linfocitos T CD8-positivos , Infecciones por VIH , Animales , Epítopos , Epítopos de Linfocito T , Macaca mulatta , Receptores de Antígenos de Linfocitos T , Factor de Necrosis Tumoral alfa
2.
PLoS Pathog ; 16(3): e1008339, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32163523

RESUMEN

Despite the success of antiretroviral therapy (ART) to halt viral replication and slow disease progression, this treatment is not curative and there remains an urgent need to develop approaches to clear the latent HIV reservoir. The human IL-15 superagonist N-803 (formerly ALT-803) is a promising anti-cancer biologic with potent immunostimulatory properties that has been extended into the field of HIV as a potential "shock and kill" therapeutic for HIV cure. However, the ability of N-803 to reactivate latent virus and modulate anti-viral immunity in vivo under the cover of ART remains undefined. Here, we show that in ART-suppressed, simian-human immunodeficiency virus (SHIV)SF162P3-infected rhesus macaques, subcutaneous administration of N-803 activates and mobilizes both NK cells and SHIV-specific CD8+ T cells from the peripheral blood to lymph node B cell follicles, a sanctuary site for latent virus that normally excludes such effector cells. We observed minimal activation of memory CD4+ T cells and no increase in viral RNA content in lymph node resident CD4+ T cells post N-803 administration. Accordingly, we found no difference in the number or magnitude of plasma viremia timepoints between treated and untreated animals during the N-803 administration period, and no difference in the size of the viral DNA cell-associated reservoir post N-803 treatment. These results substantiate N-803 as a potent immunotherapeutic candidate capable of activating and directing effector CD8+ T and NK cells to the B cell follicle during full ART suppression, and suggest N-803 must be paired with a bona fide latency reversing agent in vivo to facilitate immune-mediated modulation of the latent viral reservoir.


Asunto(s)
Antirretrovirales/administración & dosificación , Linfocitos B/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Interleucina-15/antagonistas & inhibidores , Células Asesinas Naturales/efectos de los fármacos , Proteínas/administración & dosificación , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/fisiopatología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Interleucina-15/genética , Interleucina-15/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Macaca mulatta , Proteínas Recombinantes de Fusión , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/fisiopatología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Virus de la Inmunodeficiencia de los Simios/fisiología , Latencia del Virus/efectos de los fármacos
3.
J Virol ; 93(19)2019 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-31315990

RESUMEN

Major histocompatibility complex E (MHC-E) is a highly conserved nonclassical MHC-Ib molecule that tightly binds peptides derived from leader sequences of classical MHC-Ia molecules for presentation to natural killer cells. However, MHC-E also binds diverse foreign and neoplastic self-peptide antigens for presentation to CD8+ T cells. Although the determinants of MHC-E-restricted T cell priming remain unknown, these cells are induced in humans infected with pathogens containing genes that inhibit the transporter associated with antigen processing (TAP). Indeed, mice vaccinated with TAP-inhibited autologous dendritic cells develop T cells restricted by the murine MHC-E homologue, Qa-1b. Here, we tested whether rhesus macaques (RM) vaccinated with viral constructs expressing a TAP inhibitor would develop insert-specific MHC-E-restricted CD8+ T cells. We generated viral constructs coexpressing SIVmac239 Gag in addition to one of three TAP inhibitors: herpes simplex virus 2 ICP47, bovine herpes virus 1 UL49.5, or rhesus cytomegalovirus Rh185. Each TAP inhibitor reduced surface expression of MHC-Ia molecules but did not reduce surface MHC-E expression. In agreement with modulation of surface MHC-Ia levels, TAP inhibition diminished presentation of MHC-Ia-restricted CD8+ T cell epitopes without impacting presentation of peptide antigen bound by MHC-E. Vaccination of macaques with vectors dually expressing SIVmac239 Gag with ICP47, UL49.5, or Rh185 generated Gag-specific CD8+ T cells classically restricted by MHC-Ia but not MHC-E. These data demonstrate that, in contrast to results in mice, TAP inhibition alone is insufficient for priming of MHC-E-restricted T cell responses in primates and suggest that additional unknown mechanisms govern the induction of CD8+ T cells recognizing MHC-E-bound antigen.IMPORTANCE Due to the near monomorphic nature of MHC-E in the human population and inability of many pathogens to inhibit MHC-E-mediated peptide presentation, MHC-E-restricted T cells have become an attractive vaccine target. However, little is known concerning how these cells are induced. Understanding the underlying mechanisms that induce these T cells would provide a powerful new vaccine strategy to an array of neoplasms and viral and bacterial pathogens. Recent studies have indicated a link between TAP inhibition and induction of MHC-E-restricted T cells. The significance of our research is in demonstrating that TAP inhibition alone does not prime MHC-E-restricted T cell generation and suggests that other, currently unknown mechanisms regulate their induction.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Vacunas contra el SIDAS/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Inhibidores Enzimáticos/metabolismo , Macaca mulatta , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vacunas contra el SIDAS/administración & dosificación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología
4.
J Immunol ; 200(1): 49-60, 2018 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-29150562

RESUMEN

MHC-E is a highly conserved nonclassical MHC class Ib molecule that predominantly binds and presents MHC class Ia leader sequence-derived peptides for NK cell regulation. However, MHC-E also binds pathogen-derived peptide Ags for presentation to CD8+ T cells. Given this role in adaptive immunity and its highly monomorphic nature in the human population, HLA-E is an attractive target for novel vaccine and immunotherapeutic modalities. Development of HLA-E-targeted therapies will require a physiologically relevant animal model that recapitulates HLA-E-restricted T cell biology. In this study, we investigated MHC-E immunobiology in two common nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM). Compared to humans and MCM, RM expressed a greater number of MHC-E alleles at both the population and individual level. Despite this difference, human, RM, and MCM MHC-E molecules were expressed at similar levels across immune cell subsets, equivalently upregulated by viral pathogens, and bound and presented identical peptides to CD8+ T cells. Indeed, SIV-specific, Mamu-E-restricted CD8+ T cells from RM recognized antigenic peptides presented by all MHC-E molecules tested, including cross-species recognition of human and MCM SIV-infected CD4+ T cells. Thus, MHC-E is functionally conserved among humans, RM, and MCM, and both RM and MCM represent physiologically relevant animal models of HLA-E-restricted T cell immunobiology.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad/metabolismo , Células Asesinas Naturales/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Presentación de Antígeno , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Células Cultivadas , Secuencia Conservada/genética , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Macaca fascicularis , Macaca mulatta , Modelos Animales , Péptidos/inmunología , Péptidos/metabolismo , Antígenos HLA-E
5.
Hum Gene Ther ; 34(23-24): 1204-1218, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37747811

RESUMEN

Adoptive T cell therapy using natural T cell receptor (TCR) redirection is a promising approach to fight solid cancers and viral infections in liver and other organs. However, clinical efficacy of such TCR+-T cells has been limited so far. One reason is that syngeneic preclinical models to evaluate safety and efficacy of TCR+-T cells are missing. We, therefore, developed an efficient viral vector strategy mediating expression of human major histocompatibility complex (MHC)-I in hepatocytes, which allows evaluation of TCR-T cell therapies targeting diseased liver cells. We designed adeno-associated virus (AAV) and adenoviral vectors encoding either the human-mouse chimeric HLA-A*02-like molecule, or fully human HLA-A*02 and human ß2 microglobulin (hß2m). Upon transduction of murine hepatocytes, the HLA-A*02 construct proved superior in terms of expression levels, presentation of endogenously processed peptides and activation of murine TCR+-T cells grafted with HLA-A*02-restricted, hepatitis B virus (HBV)-specific TCRs. In vivo, these T cells elicited effector function, controlled HBV replication, and reduced HBV viral load and antigen expression in livers of those mice that had received AAV-HBV and AAV-HLA-A*02. We then demonstrated the broad utility of this approach by grafting macaque T cells with the HBV-specific TCRs and enabling them to recognize HBV-infected primary macaque hepatocytes expressing HLA-A*02 upon adenoviral transduction. In conclusion, AAV and adenovirus vectors are suitable for delivery of HLA-A*02 and hß2m into mouse and macaque hepatocytes. When recognizing their cognate antigen in HLA-A*02-transduced mouse livers or on isolated macaque hepatocytes, HLA-A*02-restricted, HBV-specific TCR+-T cells become activated and exert antiviral effector functions. This approach is applicable to any MHC restriction and target disease, paving the way for safety and efficacy studies of human TCR-based therapies in physiologically relevant preclinical animal models.


Asunto(s)
Virus de la Hepatitis B , Hepatocitos , Humanos , Ratones , Animales , Virus de la Hepatitis B/genética , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T , Técnicas de Cultivo de Célula , Antígenos HLA-A
6.
J Virol ; 84(6): 2762-73, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20042498

RESUMEN

The function of plasmacytoid dendritic cells (PDC) in chronic human immunodeficiency virus type 1 (HIV-1) infection remains controversial with regard to its potential for sustained alpha interferon (IFN-alpha) production and induction of PDC-dependent tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity of HIV-infected cells. We address these areas by a study of chronically HIV-1-infected subjects followed through antiretroviral therapy (ART) interruption and by testing PDC cytolytic function against autologous HIV-infected CD4(+) T cells. Rebound in viremia induced by therapy interruption showed a positive association between TRAIL and viral load or T-cell activation, but comparable levels of plasma IFN-alpha/beta were found in viremic ART-treated and control subjects. While PDC from HIV-infected subjects expressed less interferon regulator factor 7 (IRF-7) and produced significantly less IFN-alpha upon Toll-like receptor 7/9 (TLR7/9) engagement than controls, membrane TRAIL expression in PDC from HIV(+) subjects was increased. Moreover, no significant increase in death receptor 5 (DR5) expression was seen in CD4(+) T cells from viremic HIV(+) subjects compared to controls or following in vitro infection/exposure to infectious and noninfectious virus or exogenous IFN-alpha, respectively. Although activated PDC killed the DR5-expressing HIV-infected Sup-T1 cell line, PDC did not lyse primary autologous HIV(+) CD4(+) T cells yet could provide accessory help for NK cells in killing HIV-infected autologous CD4(+) T cells. Taken together, our data show a lack of sustained high levels of soluble IFN-alpha in chronic HIV-1 infection in vivo and document a lack of direct PDC cytolytic activity against autologous infected or uninfected CD4(+) T cells.


Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Células Dendríticas/metabolismo , Infecciones por VIH/inmunología , Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Animales , Linfocitos T CD4-Positivos/citología , Línea Celular , Células Dendríticas/citología , Femenino , VIH-1/inmunología , Humanos , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Interferón-alfa/inmunología , Células Asesinas Naturales/inmunología , Masculino , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Carga Viral , Viremia/inmunología , Viremia/virología
7.
Sci Immunol ; 6(57)2021 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-33766848

RESUMEN

Human leukocyte antigen-E (HLA-E) normally presents an HLA class Ia signal peptide to the NKG2A/C-CD94 regulatory receptors on natural killer (NK) cells and T cell subsets. Rhesus macaques immunized with a cytomegalovirus-vectored simian immunodeficiency virus (SIV) vaccine generated Mamu-E (HLA-E homolog)-restricted T cell responses that mediated post-challenge SIV replication arrest in >50% of animals. However, HIV-1-specific, HLA-E-restricted T cells have not been observed in HIV-1-infected individuals. Here, HLA-E-restricted, HIV-1-specific CD8 + T cells were primed in vitro. These T cell clones and allogeneic CD8 + T cells transduced with their T cell receptors suppressed HIV-1 replication in CD4 + T cells in vitro. Vaccine induction of efficacious HLA-E-restricted HIV-1-specific T cells should therefore be possible.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Interacciones Huésped-Patógeno/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunología , Secuencia de Aminoácidos , Biomarcadores , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Citocinas/metabolismo , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Infecciones por VIH/metabolismo , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , Humanos , Inmunofenotipificación , Células Jurkat , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Péptidos/química , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Antígenos HLA-E
8.
Science ; 372(6541)2021 04 30.
Artículo en Inglés | MEDLINE | ID: mdl-33766941

RESUMEN

Strain 68-1 rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens elicit CD8+ T cells recognizing epitopes presented by major histocompatibility complex II (MHC-II) and MHC-E but not MHC-Ia. These immune responses mediate replication arrest of SIV in 50 to 60% of monkeys. We show that the peptide VMAPRTLLL (VL9) embedded within the RhCMV protein Rh67 promotes intracellular MHC-E transport and recognition of RhCMV-infected fibroblasts by MHC-E-restricted CD8+ T cells. Deletion or mutation of viral VL9 abrogated MHC-E-restricted CD8+ T cell priming, resulting in CD8+ T cell responses exclusively targeting MHC-II-restricted epitopes. These responses were comparable in magnitude and differentiation to responses elicited by 68-1 vectors but did not protect against SIV. Thus, Rh67-enabled direct priming of MHC-E-restricted T cells is crucial for RhCMV/SIV vaccine efficacy.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Citomegalovirus/metabolismo , Vectores Genéticos/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Fragmentos de Péptidos/metabolismo , Vacunas contra el SIDAS/inmunología , Animales , Línea Celular , Citomegalovirus/genética , Epítopos de Linfocito T/inmunología , Fibroblastos/metabolismo , Vectores Genéticos/genética , Antígenos de Histocompatibilidad Clase I/genética , Ligandos , Macaca mulatta , Fragmentos de Péptidos/genética , Transporte de Proteínas , Virus de la Inmunodeficiencia de los Simios , Antígenos HLA-E
9.
Nat Commun ; 12(1): 3343, 2021 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-34099693

RESUMEN

In the absence of a prophylactic vaccine, the use of antiretroviral therapy (ART) as pre-exposure prophylaxis (PrEP) to prevent HIV acquisition by uninfected individuals is a promising approach to slowing the epidemic, but its efficacy is hampered by incomplete patient adherence and ART-resistant variants. Here, we report that competitive inhibition of HIV Env-CCR5 binding via the CCR5-specific antibody Leronlimab protects rhesus macaques against infection following repeated intrarectal challenges of CCR5-tropic SHIVSF162P3. Injection of Leronlimab weekly at 10 mg/kg provides significant but partial protection, while biweekly 50 mg/kg provides complete protection from SHIV acquisition. Tissue biopsies from protected macaques post challenge show complete CCR5 receptor occupancy and an absence of viral nucleic acids. After Leronlimab washout, protected macaques remain aviremic, and adoptive transfer of hematologic cells into naïve macaques does not transmit viral infection. These data identify CCR5 blockade with Leronlimab as a promising approach to HIV prophylaxis and support initiation of clinical trials.


Asunto(s)
Receptores CCR5/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/transmisión , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Femenino , Anticuerpos Anti-VIH/farmacología , Infecciones por VIH , Humanos , Macaca mulatta , Masculino , Membrana Mucosa , Profilaxis Pre-Exposición , Carga Viral
10.
Nat Commun ; 10(1): 3753, 2019 08 21.
Artículo en Inglés | MEDLINE | ID: mdl-31434895

RESUMEN

Semen is the vehicle for virion dissemination in the female reproductive tract (FRT) in male-to-female HIV transmission. Recent data suggests that higher frequency semen exposure is associated with activation of anti-HIV mechanisms in HIV negative sex workers. Here, we use a non-human primate (NHP) model to show that repeated vaginal exposure to semen significantly reduces subsequent infection by repeated low-dose vaginal SIVmac251 challenge. Repeated semen exposures result in lower CCR5 expression in circulating CD4+ T-cells, as well as higher expression of Mx1 (in correlation with IFNε expression) and FoxP3 in the cervicovaginal mucosa, and increased infiltration of CD4+ T-cells. Establishing in vivo evidence of competing effects of semen on transmission impacts our basic understanding of what factors may determine HIV infectivity in humans. Our results clearly indicate that repeated semen exposure can profoundly modulate the FRT microenvironment, paradoxically promoting host resistance against HIV acquisition.


Asunto(s)
Cuello del Útero/inmunología , Membrana Mucosa/inmunología , Semen/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/transmisión , Virus de la Inmunodeficiencia de los Simios/inmunología , Vagina/inmunología , Animales , Linfocitos T CD4-Positivos , Cuello del Útero/virología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Factores de Transcripción Forkhead/metabolismo , Infecciones por VIH/inmunología , Infecciones por VIH/transmisión , Humanos , Macaca mulatta , Membrana Mucosa/metabolismo , Proteínas de Resistencia a Mixovirus/metabolismo , Receptores CCR5/metabolismo , Vagina/virología
11.
Immunol Res ; 42(1-3): 219-32, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-19066740

RESUMEN

We have focused our research on understanding the basic biology of and developing novel therapeutic and prophylactic DNA vaccines. We have among others three distinct primary areas of interest which include: 1. Enhancing in vivo delivery and transfection of DNA vaccine vectors 2. Improving DNA vaccine construct immunogenicity 3. Using molecular adjuvants to modulate and skew immune responses. Key to the immunogenicity of DNA vaccines is the presentation of expressed antigen to antigen-presenting cells. To improve expression and presentation of antigen, we have investigated various immunization methods with current focus on a combination of intramuscular injection and electroporation. To improve our vaccine constructs, we also employed methods such as RNA/codon optimization and antigen consensus to enhance expression and cellular/humoral cross-reactivity, respectively. Our lab also researches the potential of various molecular adjuvants to skew Th1/Th2 responses, enhance cellular/humoral responses, and improve protection in various animal models. Through improving our understanding of basic immunology as it is related to DNA vaccine technology, our goal is to develop the technology to the point of utility for human and animal health.


Asunto(s)
Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Virosis/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/uso terapéutico , Animales , Vectores Genéticos/genética , Humanos , Transfección/métodos , Vacunas de ADN/genética , Vacunas de ADN/uso terapéutico , Vacunas Virales/genética , Virosis/prevención & control , Virosis/terapia , Virus/genética , Virus/inmunología
12.
Nat Commun ; 9(1): 4833, 2018 11 13.
Artículo en Inglés | MEDLINE | ID: mdl-30420666

RESUMEN

The original version of this Article contained an error in the spelling of the author Jonah B Sacha, which was incorrectly given as Jonah Sacha. These errors have now been corrected in both the PDF and HTML versions of the Article.

13.
Nat Commun ; 9(1): 3137, 2018 08 07.
Artículo en Inglés | MEDLINE | ID: mdl-30087334

RESUMEN

Through major histocompatibility complex class Ia leader sequence-derived (VL9) peptide binding and CD94/NKG2 receptor engagement, human leucocyte antigen E (HLA-E) reports cellular health to NK cells. Previous studies demonstrated a strong bias for VL9 binding by HLA-E, a preference subsequently supported by structural analyses. However, Mycobacteria tuberculosis (Mtb) infection and Rhesus cytomegalovirus-vectored SIV vaccinations revealed contexts where HLA-E and the rhesus homologue, Mamu-E, presented diverse pathogen-derived peptides to CD8+ T cells, respectively. Here we present crystal structures of HLA-E in complex with HIV and Mtb-derived peptides. We show that despite the presence of preferred primary anchor residues, HLA-E-bound peptides can adopt alternative conformations within the peptide binding groove. Furthermore, combined structural and mutagenesis analyses illustrate a greater tolerance for hydrophobic and polar residues in the primary pockets than previously appreciated. Finally, biochemical studies reveal HLA-E peptide binding and exchange characteristics with potential relevance to its alternative antigen presenting function in vivo.


Asunto(s)
Epítopos , Antígenos de Histocompatibilidad Clase I/inmunología , Péptidos/inmunología , Animales , Presentación de Antígeno , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Citomegalovirus , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Humanos , Células Asesinas Naturales/inmunología , Macaca mulatta , Mycobacterium tuberculosis , Unión Proteica , Conformación Proteica , Virus de la Inmunodeficiencia de los Simios/inmunología , Antígenos HLA-E
14.
PLoS One ; 12(3): e0170306, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28278250

RESUMEN

Human semen has the potential to modulate the epithelial mucosal tissues it contacts, as seminal plasma (SP) is recognized to contain both pro- and anti-barrier components, yet its effects on epithelial barrier function are largely unknown. We addressed the role of human SP when exposed to the basal-lateral epithelial surface, a situation that would occur clinically with prior mechanical or disease-related injury of the human epithelial mucosal cell layers in contact with semen. The action of SP on claudins-2, -4, -5, and -7 expression, as well as on a target epithelium whose basolateral surface has been made accessible to SP, showed upregulation of claudins-4 and -5 in CACO-2 human epithelial cell layers, despite broad variance in SP-induced modulation of transepithelial electrical resistance and mannitol permeability. Upregulation of claudin-2 by SP also exhibited such variance by SP sample. We characterize individual effects on CACO-2 barrier function of nine factors known to be present abundantly in seminal plasma (zinc, EGF, citrate, spermine, fructose, urea, TGF, histone, inflammatory cytokines) to establish that zinc, spermine and fructose had significant potential to raise CACO-2 transepithelial resistance, whereas inflammatory cytokines and EGF decreased this measure of barrier function. The role of zinc as a dominant factor in determining higher levels of transepithelial resistance and lower levels of paracellular leak were confirmed by zinc chelation and exogenous zinc addition. As expected, SP presentation to the basolateral cell surface also caused a very dramatic yet transient elevation of pErk levels. Results suggest that increased zinc content in SP can compete against the barrier-compromising effect of negative modulators in SP when SP gains access to that epithelium's basolateral surface. Prophylactic elevation of zinc in an epithelial cell layer prior to contact by SP may help to protect an epithelial barrier from invasion by SP-containing STD microbial pathogens such as HPV or HIV.


Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Epitelio/fisiología , Semen/química , Zinc/farmacología , Células CACO-2 , Claudinas/metabolismo , Citocinas/metabolismo , Epitelio/efectos de los fármacos , Humanos , Masculino
15.
Nat Commun ; 8(1): 2146, 2017 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-29247188

RESUMEN

Hepatitis B virus (HBV) is a major global health concern, and the development of curative therapeutics is urgently needed. Such efforts are impeded by the lack of a physiologically relevant, pre-clinical animal model of HBV infection. Here, we report that expression of the HBV entry receptor, human sodium-taurocholate cotransporting polypeptide (hNTCP), on macaque primary hepatocytes facilitates HBV infection in vitro, where all replicative intermediates including covalently closed circular DNA (cccDNA) are present. Furthermore, viral vector-mediated expression of hNTCP on hepatocytes in vivo renders rhesus macaques permissive to HBV infection. These in vivo macaque HBV infections are characterized by longitudinal HBV DNA in serum, and detection of HBV DNA, RNA, and HBV core antigen (HBcAg) in hepatocytes. Together, these results show that expressing hNTCP on macaque hepatocytes renders them susceptible to HBV infection, thereby establishing a physiologically relevant model of HBV infection to study immune clearance and test therapeutic and curative approaches.


Asunto(s)
Virus de la Hepatitis B/fisiología , Hepatocitos/metabolismo , Hepatocitos/virología , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismo , Animales , Células Cultivadas , ADN Viral/metabolismo , Hepatitis B/genética , Hepatitis B/metabolismo , Hepatitis B/virología , Antígenos del Núcleo de la Hepatitis B/metabolismo , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/metabolismo , Hepatocitos/citología , Interacciones Huésped-Patógeno , Humanos , Macaca mulatta , Transportadores de Anión Orgánico Sodio-Dependiente/genética , ARN Viral/metabolismo , Simportadores/genética
16.
Nat Commun ; 8(1): 1418, 2017 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-29127275

RESUMEN

Allogeneic hematopoietic stem cell transplantation (HSCT) is a critically important therapy for hematological malignancies, inborn errors of metabolism, and immunodeficiency disorders, yet complications such as graft-vs.-host disease (GvHD) limit survival. Development of anti-GvHD therapies that do not adversely affect susceptibility to infection or graft-vs.-tumor immunity are hampered by the lack of a physiologically relevant, preclinical model of allogeneic HSCT. Here we show a spectrum of diverse clinical HSCT outcomes including primary and secondary graft failure, lethal GvHD, and stable, disease-free full donor engraftment using reduced intensity conditioning and mobilized peripheral blood HSCT in unrelated, fully MHC-matched Mauritian-origin cynomolgus macaques. Anti-GvHD prophylaxis of tacrolimus, post-transplant cyclophosphamide, and CD28 blockade induces multi-lineage, full donor chimerism and recipient-specific tolerance while maintaining pathogen-specific immunity. These results establish a new preclinical allogeneic HSCT model for evaluation of GvHD prophylaxis and next-generation HSCT-mediated therapies for solid organ tolerance, cure of non-malignant hematological disease, and HIV reservoir clearance.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas/métodos , Macaca fascicularis/inmunología , Complejo Mayor de Histocompatibilidad , Animales , Femenino , Enfermedad Injerto contra Huésped/prevención & control , Prueba de Histocompatibilidad , Humanos , Macaca fascicularis/genética , Masculino , Modelos Animales , Especificidad de la Especie , Quimera por Trasplante/genética , Quimera por Trasplante/inmunología , Tolerancia al Trasplante/genética , Tolerancia al Trasplante/inmunología , Trasplante Homólogo , Resultado del Tratamiento
17.
J Acquir Immune Defic Syndr ; 65(4): 405-13, 2014 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-24226059

RESUMEN

OBJECTIVE: Intravaginal exposure to simian immunodeficiency virus (SIV) acutely recruits interferon-alpha (IFN-α) producing plasmacytoid dendritic cells (pDC) and CD4 T-lymphocyte targets to the endocervix of nonhuman primates. We tested the impact of repeated cervicovaginal exposures to noninfectious, defective SIV particles over 72 hours on a subsequent cervicovaginal challenge with replication competent SIV. METHODS: Thirty-four female Indian Rhesus macaques were given a 3-day twice-daily vaginal exposures to either SIVsmB7, a replication-deficient derivative of SIVsmH3 produced by a T lymphoblast CEMx174 cell clone (n = 16), or to CEM supernatant controls (n = 18). On the fourth day, animals were either euthanized to assess cervicovaginal immune cell infiltration or intravaginally challenged with SIVmac251. Challenged animals were tracked for plasma viral load and CD4 counts and euthanized at 42 days after infection. RESULTS: At the time of challenge, macaques exposed to SIVsmB7, had higher levels of cervical CD123 pDCs (P = 0.032) and CD4 T cells (P = 0.036) than those exposed to CEM control. Vaginal tissues showed a significant increase in CD4 T-cell infiltrates (P = 0.048) and a trend toward increased CD68 cellular infiltrates. After challenge, 12 SIVsmB7-treated macaques showed 2.5-fold greater daily rate of CD4 decline (P = 0.0408), and viral load rise (P = 0.0036) as compared with 12 control animals. CONCLUSIONS: Repeated nonproductive exposure to viral particles within a short daily time frame did not protect against infection despite pDC recruitment, resulting instead in an accelerated CD4 T-cell loss with an increased rate of viral replication.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Cuello del Útero/inmunología , Células Dendríticas/inmunología , Endometrio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Vagina/inmunología , Animales , Recuento de Linfocito CD4 , Cuello del Útero/virología , Endometrio/virología , Femenino , Macaca mulatta , Plasma/virología , Vagina/virología , Carga Viral
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