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2.
J Immunol ; 208(2): 429-443, 2022 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-34903642

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces T cell, B cell, and Ab responses that are detected for several months in recovered individuals. Whether this response resembles a typical respiratory viral infection is a matter of debate. In this study, we followed T cell and Ab responses in 24 mainly nonhospitalized human subjects who had recovered from PCR-confirmed SARS-CoV-2 infection at two time points (median of 45 and 145 d after symptom onset). Ab responses were detected in 95% of subjects, with a strong correlation between plasma and salivary anti-spike (anti-S) and anti-receptor binding domain IgG, as well as a correlation between circulating T follicular helper cells and the SARS-CoV-2-specific IgG response. T cell responses to SARS-CoV-2 peptides were determined using intracellular cytokine staining, activation markers, proliferation, and cytokine secretion. All study subjects had a T cell response to at least one SARS-CoV-2 Ag based on at least one T cell assay. CD4+ responses were largely of the Th1 phenotype, but with a lower ratio of IFN-γ- to IL-2-producing cells and a lower frequency of CD8+:CD4+ T cells than in influenza A virus (IAV)-specific memory responses within the same subjects. Analysis of secreted molecules also revealed a lower ratio of IFN-γ to IL-2 and an altered cytotoxic profile for SARS-CoV-2 S- and nucleocapsid-specific responses compared with IAV-specific responses. These data suggest that the memory T cell phenotype after a single infection with SARS-CoV-2 persists over time, with an altered cytokine and cytotoxicity profile compared with long-term memory to whole IAV within the same subjects.


Asunto(s)
Formación de Anticuerpos , COVID-19/inmunología , Inmunidad Celular , Inmunoglobulina G/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Células TH1/inmunología , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de Tiempo
3.
J Immunol ; 206(1): 37-50, 2021 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-33208459

RESUMEN

There is a pressing need for an in-depth understanding of immunity to SARS-CoV-2. In this study, we investigated human T cell recall responses to fully glycosylated spike trimer, recombinant N protein, as well as to S, N, M, and E peptide pools in the early convalescent phase and compared them with influenza-specific memory responses from the same donors. All subjects showed SARS-CoV-2-specific T cell responses to at least one Ag. Both SARS-CoV-2-specific and influenza-specific CD4+ T cell responses were predominantly of the central memory phenotype; however SARS-CoV-2-specific CD4+ T cells exhibited a lower IFN-γ to TNF ratio compared with influenza-specific memory responses from the same donors, independent of disease severity. SARS-CoV-2-specific T cells were less multifunctional than influenza-specific T cells, particularly in severe cases, potentially suggesting exhaustion. Most SARS-CoV-2-convalescent subjects also produced IFN-γ in response to seasonal OC43 S protein. We observed granzyme B+/IFN-γ+, CD4+, and CD8+ proliferative responses to peptide pools in most individuals, with CD4+ T cell responses predominating over CD8+ T cell responses. Peripheral T follicular helper (pTfh) responses to S or N strongly correlated with serum neutralization assays as well as receptor binding domain-specific IgA; however, the frequency of pTfh responses to SARS-CoV-2 was lower than the frequency of pTfh responses to influenza virus. Overall, T cell responses to SARS-CoV-2 are robust; however, CD4+ Th1 responses predominate over CD8+ T cell responses, have a more inflammatory profile, and have a weaker pTfh response than the response to influenza virus within the same donors, potentially contributing to COVID-19 disease.


Asunto(s)
Antígenos Virales/inmunología , Linfocitos T CD4-Positivos/inmunología , Inflamación/inmunología , Orthomyxoviridae/inmunología , SARS-CoV-2/inmunología , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad
4.
Nature ; 517(7534): 386-90, 2015 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-25363763

RESUMEN

T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.


Asunto(s)
Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Tolerancia Inmunológica/inmunología , Proteínas de la Membrana/metabolismo , Receptores Virales/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Antígenos CD/química , Antígenos CD/inmunología , Autoinmunidad/inmunología , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/inmunología , Línea Celular , Neoplasias Colorrectales/inmunología , Modelos Animales de Enfermedad , Femenino , Receptor 2 Celular del Virus de la Hepatitis A , Humanos , Inflamación/inmunología , Inflamación/patología , Ligandos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Moleculares , Membrana Mucosa/inmunología , Membrana Mucosa/patología , Conformación Proteica , Multimerización de Proteína , Receptores Virales/química , Receptores Virales/inmunología
5.
PLoS Comput Biol ; 15(9): e1006453, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31568525

RESUMEN

Characterization of Human Endogenous Retrovirus (HERV) expression within the transcriptomic landscape using RNA-seq is complicated by uncertainty in fragment assignment because of sequence similarity. We present Telescope, a computational software tool that provides accurate estimation of transposable element expression (retrotranscriptome) resolved to specific genomic locations. Telescope directly addresses uncertainty in fragment assignment by reassigning ambiguously mapped fragments to the most probable source transcript as determined within a Bayesian statistical model. We demonstrate the utility of our approach through single locus analysis of HERV expression in 13 ENCODE cell types. When examined at this resolution, we find that the magnitude and breadth of the retrotranscriptome can be vastly different among cell types. Furthermore, our approach is robust to differences in sequencing technology and demonstrates that the retrotranscriptome has potential to be used for cell type identification. We compared our tool with other approaches for quantifying transposable element (TE) expression, and found that Telescope has the greatest resolution, as it estimates expression at specific TE insertions rather than at the TE subfamily level. Telescope performs highly accurate quantification of the retrotranscriptomic landscape in RNA-seq experiments, revealing a differential complexity in the transposable element biology of complex systems not previously observed. Telescope is available at https://github.com/mlbendall/telescope.


Asunto(s)
Elementos Transponibles de ADN/genética , Retrovirus Endógenos/genética , Perfilación de la Expresión Génica/métodos , Programas Informáticos , Transcriptoma/genética , Línea Celular , Biología Computacional , Técnicas Citológicas , Humanos , Especificidad de Órganos , Análisis de Secuencia de ARN/métodos
6.
J Virol ; 92(19)2018 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-29997203

RESUMEN

Human immunodeficiency virus type 1 (HIV-1) infection often arises from a single transmitted/founder (TF) viral variant among a large pool of viruses in the quasispecies in the transmitting partner. TF variants are typically nondominant in blood and genital secretions, indicating that they have unique traits. The plasmacytoid dendritic cell (pDC) is the primary alpha interferon (IFN-α)-producing cell in response to viral infections and is rapidly recruited to the female genital tract upon exposure to HIV-1. The impact of pDCs on transmission is unknown. We investigated whether evasion of pDC responses is a trait of TF viruses. pDCs from healthy donors were stimulated in vitro with a panel of 20 HIV-1 variants, consisting of one TF variant and three nontransmitted (NT) variants each from five transmission-linked donor pairs, and secretion of IFN-α and tumor necrosis factor alpha (TNF-α) was measured by enzyme-linked immunosorbent assay (ELISA). No significant differences in cytokine secretion in response to TF and NT viruses were observed, despite a trend toward enhanced IFN-α and TNF-α production in response to TF viruses. NT viruses demonstrated polarization toward production of either IFN-α or TNF-α, indicating possible dysregulation. Also, for NT viruses, IFN-α secretion was associated with increased resistance of the virus to inactivation by IFN-α in vitro, suggesting in vivo evolution. Thus, TF viruses do not appear to preferentially subvert pDC activation compared to that with nontransmitted HIV-1 variants. pDCs may, however, contribute to the in vivo evolution of HIV-1.IMPORTANCE The plasmacytoid dendritic cell (pDC) is the first cell type recruited to the site of HIV-1 exposure; however, its contribution to the viral bottleneck in HIV-1 transmission has not been explored previously. We hypothesized that transmitted/founder viruses are able to avoid the pDC response. In this study, we used previously established donor pair-linked transmitted/founder and nontransmitted (or chronic) variants of HIV-1 to stimulate pDCs. Transmitted/founder HIV-1, instead of suppressing pDC responses, induced IFN-α and TNF-α secretion to levels comparable to those induced by viruses from the transmitting partner. We noted several unique traits of chronic viruses, including polarization between IFN-α and TNF-α production as well as a strong relationship between IFN-α secretion and the resistance of the virus to neutralization. These data rule out the possibility that TF viruses preferentially suppress pDCs in comparison to the pDC response to nontransmitted HIV variants. pDCs may, however, be important drivers of viral evolution in vivo.


Asunto(s)
Células Dendríticas/inmunología , Infecciones por VIH/transmisión , VIH-1/inmunología , Interferón-alfa/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Células Dendríticas/virología , Femenino , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/patogenicidad , Interacciones Huésped-Patógeno , Humanos , Interferón-alfa/inmunología , Masculino , Pruebas de Neutralización , Cultivo Primario de Células , Factor de Necrosis Tumoral alfa/inmunología , Virión/inmunología , Virión/patogenicidad
7.
J Immunol ; 198(8): 3181-3194, 2017 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-28264968

RESUMEN

In chronic diseases, such as HIV infection, plasmacytoid dendritic cells (pDCs) are rendered dysfunctional, as measured by their decreased capacity to produce IFN-α. In this study, we identified elevated levels of T cell Ig and mucin-domain containing molecule-3 (Tim-3)-expressing pDCs in the blood of HIV-infected donors. The frequency of Tim-3-expressing pDCs correlated inversely with CD4 T cell counts and positively with HIV viral loads. A lower frequency of pDCs expressing Tim-3 produced IFN-α or TNF-α in response to the TLR7 agonists imiquimod and Sendai virus and to the TLR9 agonist CpG. Thus, Tim-3 may serve as a biomarker of pDC dysfunction in HIV infection. The source and function of Tim-3 was investigated on enriched pDC populations from donors not infected with HIV. Tim-3 induction was achieved in response to viral and artificial stimuli, as well as exogenous IFN-α, and was PI3K dependent. Potent pDC-activating stimuli, such as CpG, imiquimod, and Sendai virus, induced the most Tim-3 expression and subsequent dysfunction. Small interfering RNA knockdown of Tim-3 increased IFN-α secretion in response to activation. Intracellular Tim-3, as measured by confocal microscopy, was dispersed throughout the cytoplasm prior to activation. Postactivation, Tim-3 accumulated at the plasma membrane and associated with disrupted TLR9 at the submembrane. Tim-3-expressing pDCs had reduced IRF7 levels. Furthermore, intracellular Tim-3 colocalized with p85 and IRF7 within LAMP1+ lysosomes, suggestive of a role in degradation. We conclude that Tim-3 is a biomarker of dysfunctional pDCs and may negatively regulate IFN-α, possibly through interference with TLR signaling and recruitment of IRF7 and p85 into lysosomes, enhancing their degradation.


Asunto(s)
Biomarcadores/análisis , Células Dendríticas/inmunología , Infecciones por VIH/inmunología , Receptor 2 Celular del Virus de la Hepatitis A/inmunología , Transducción de Señal/inmunología , Adulto , Separación Celular , Células Dendríticas/metabolismo , Femenino , Infecciones por VIH/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Humanos , Factor 7 Regulador del Interferón/inmunología , Factor 7 Regulador del Interferón/metabolismo , Lisosomas/inmunología , Lisosomas/metabolismo , Masculino , Microscopía Confocal , Persona de Mediana Edad , Receptor Toll-Like 9/inmunología , Receptor Toll-Like 9/metabolismo , Adulto Joven
8.
J Infect Dis ; 218(8): 1210-1218, 2018 09 08.
Artículo en Inglés | MEDLINE | ID: mdl-29800309

RESUMEN

Background: Attenuated varicella zoster virus (VZV) is a promising vector for recombinant vaccines. Because human immunodeficiencyvirus (HIV) vaccines are believed to require mucosal immunogenicity, we characterized mucosal VZV-specific humoral immunity following VZVOka vaccination. Methods: Adult Kenyan VZV-seropositive women (n = 44) received a single dose of the live zoster VZVOka vaccine. The anamnestic responses to the virus were followed longitudinally in both plasma and mucosal secretions using an in-house glycoprotein enzyme-linked immunosorbent assay and safety and reactogenicity monitored. VZV seroprevalence and baseline responses to the virus were also characterized in our cohorts (n = 288). Results: Besides boosting anti-VZV antibody responses systemically, vaccination also boosted anti-VZV immunity in the cervicovaginal mucosa with a 2.9-fold rise in immunoglobulin G (P < .0001) and 1.6-fold rise in immunoglobulin A (IgA) (P = .004) from the time before immunization and 4 weeks postvaccination. Baseline analysis demonstrated high avidity antibodies at the gastrointestinal and genital mucosa of VZV-seropositive women. Measurement of VZV-specific IgA in saliva is a sensitive tool for detecting prior VZV infection. Conclusions: VZVOka vaccine was safe and immunogenic in VZV-seropositive adult Kenyan women. We provided compelling evidence of VZV ability to induce genital mucosa immunity. Clinical Trials Registration: NCT02514018.


Asunto(s)
Anticuerpos Antivirales/metabolismo , Herpesvirus Humano 3/aislamiento & purificación , Inmunidad Humoral , Membrana Mucosa/inmunología , Vagina/inmunología , Infección por el Virus de la Varicela-Zóster/prevención & control , Anticuerpos Antivirales/sangre , Femenino , Vacuna contra el Herpes Zóster/inmunología , Humanos , Kenia/epidemiología , Vacunas Atenuadas , Infección por el Virus de la Varicela-Zóster/epidemiología , Infección por el Virus de la Varicela-Zóster/inmunología
9.
J Virol ; 91(16)2017 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-28592534

RESUMEN

Immunotherapy with passive administration of broadly neutralizing HIV-1 envelope-specific antibodies (bnAbs) in the setting of established infection in vivo has yielded mixed results. The contribution of different antibodies toward the direct elimination of infected cells is poorly understood. In this study, we determined the ability of 12 well-characterized anti-HIV-1 neutralizing antibodies to recognize and eliminate primary CD4 T cells infected with HIV-1 belonging to clades A, B, C, and D, via antibody-dependent complement-mediated lysis (ADCML) and antibody-dependent cell-mediated cytotoxicity (ADCC), in vitro We further tested unique combinations of these antibodies to determine the optimal antibody cocktails to be tested in future clinical trials. We report that antibody binding to infected CD4 T cells is highly variable and correlates with ADCML and ADCC processes. Particularly, antibodies targeting the envelope glycan shield (2G12) and V1/V2 site (PG9, PG16, and PGT145) are best at recognizing HIV-1-infected CD4 T cells. However, only PG9 and PG16 and their combinations with other bnAbs sufficiently induced the elimination of HIV-1-infected CD4 T cells by ADCML, ADCC, or both. Notably, CD4 binding site antibodies VRC01, 3BNC117, and NIH45-46 G54W did not exhibit recognition of infected cells and were unable to induce their killing. Future trials geared toward the development of a cure for HIV/AIDS should incorporate V1/V2 antibodies for maximal clearance of infected cells. With the use of only primary immune cells, we conducted a comprehensive cross-clade physiological analysis to aid the direction of antibodies as therapeutics toward the development of a cure for HIV/AIDS.IMPORTANCE Several antibodies capable of neutralizing the majority of circulating HIV-1 strains have been identified to date and have been shown to prevent infection in animal models. However, the use of combinations of such broadly neutralizing antibodies (bnAbs) for the treatment and eradication of HIV-1 in infected humans remains uncertain. In this study, we tested the ability of bnAbs to directly recognize and eliminate primary human CD4 T cells infected with diverse HIV-1 strains representative of the global epidemic by antibody-dependent pathways. We also tested several combinations of bnAbs in our assays in order to maximize the clearance of infected cells. We show that the ability of bnAbs to identify and kill infected cells is highly variable and that only a few of them are able to exert this function. Our data will help guide the formulation of bnAbs to test in future human trials aimed at the development of a cure.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Proteínas del Sistema Complemento/inmunología , Reacciones Cruzadas , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Humanos
10.
PLoS Pathog ; 12(4): e1005545, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-27082643

RESUMEN

Resting CD4+ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8+ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8+ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8+ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8+ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8+ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam3CSK4. In contrast, we did not observe CD8+ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist 'ALT-803', an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8+ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8+ T-cells in HIV eradication strategies.


Asunto(s)
Antivirales/farmacología , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/inmunología , Proteínas/farmacología , Linfocitos T Citotóxicos/inmunología , Latencia del Virus/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Ensayo de Immunospot Ligado a Enzimas , Citometría de Flujo , Humanos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión , Activación Viral/efectos de los fármacos
11.
Nat Mater ; 15(11): 1212-1221, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27525571

RESUMEN

The liver and spleen are major biological barriers to translating nanomedicines because they sequester the majority of administered nanomaterials and prevent delivery to diseased tissue. Here we examined the blood clearance mechanism of administered hard nanomaterials in relation to blood flow dynamics, organ microarchitecture and cellular phenotype. We found that nanomaterial velocity reduces 1,000-fold as they enter and traverse the liver, leading to 7.5 times more nanomaterial interaction with hepatic cells relative to peripheral cells. In the liver, Kupffer cells (84.8 ± 6.4%), hepatic B cells (81.5 ± 9.3%) and liver sinusoidal endothelial cells (64.6 ± 13.7%) interacted with administered PEGylated quantum dots, but splenic macrophages took up less material (25.4 ± 10.1%) due to differences in phenotype. The uptake patterns were similar for two other nanomaterial types and five different surface chemistries. Potential new strategies to overcome off-target nanomaterial accumulation may involve manipulating intra-organ flow dynamics and modulating the cellular phenotype to alter hepatic cell interactions.


Asunto(s)
Hígado/metabolismo , Nanoestructuras , Dureza , Hígado/citología , Fenotipo , Propiedades de Superficie
13.
J Virol ; 89(7): 3723-36, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25609823

RESUMEN

UNLABELLED: Chronic HIV infection results in a loss of HIV-specific CD8(+) T cell effector function, termed "exhaustion," which is mediated, in part, by the membrane coinhibitory receptor T cell immunoglobulin mucin domain-3 (Tim-3). Like many other receptors, a soluble form of this protein has been described in human blood plasma. However, soluble Tim-3 (sTim-3) is poorly characterized, and its role in HIV disease is unknown. Here, we show that Tim-3 is shed from the surface of responding CD8(+) T cells by the matrix metalloproteinase ADAM10, producing a soluble form of the coinhibitory receptor. Despite previous reports in the mouse model, no alternatively spliced, soluble form of Tim-3 was observed in humans. Shed sTim-3 was found in human plasma and was significantly elevated during early and chronic untreated HIV infection, but it was not found differentially modulated in highly active antiretroviral therapy (HAART)-treated HIV-infected subjects or in elite controllers compared to HIV-uninfected subjects. Plasma sTim-3 levels were positively correlated with HIV load and negatively correlated with CD4 counts. Thus, plasma sTim-3 shedding correlated with HIV disease progression. Despite these correlations, we found that shedding Tim-3 did not improve the function of CD8(+) T cells in terms of gamma interferon production or prevent their apoptosis through galectin-9. Further characterization studies of sTim-3 function are needed to understand the contribution of sTim-3 in HIV disease pathogenesis, with implications for novel therapeutic interventions. IMPORTANCE: Despite the overall success of HAART in slowing the progression to AIDS in HIV-infected subjects, chronic immune activation and T cell exhaustion contribute to the eventual deterioration of the immune system. Understanding these processes will aid in the development of interventions and therapeutics to be used in combination with HAART to slow or reverse this deterioration. Here, we show that a soluble form of T cell exhaustion associated coinhibitory molecule 3, sTim-3, is shed from the surface of T cells. Furthermore, sTim-3 is elevated in the plasma of treatment-naive subjects with acute or chronic HIV infection and is associated with markers of disease progression. This is the first study to characterize sTim-3 in human plasma, its source, and mechanism of production. While it is still unclear whether sTim-3 contributes to HIV pathogenesis, sTim-3 may represent a new correlate of HIV disease progression.


Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Biomarcadores/sangre , Linfocitos T CD8-positivos/metabolismo , Infecciones por VIH/diagnóstico , Infecciones por VIH/patología , Proteínas de la Membrana/sangre , Proteínas de la Membrana/metabolismo , Plasma/química , Proteína ADAM10 , Recuento de Linfocito CD4 , Estudios de Cohortes , Progresión de la Enfermedad , Receptor 2 Celular del Virus de la Hepatitis A , Humanos , Estudios Prospectivos , Carga Viral
14.
PLoS Pathog ; 10(8): e1004287, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25122219

RESUMEN

Resting memory CD4+ T-cells harboring latent HIV proviruses represent a critical barrier to viral eradication. Histone deacetylase inhibitors (HDACis), such as suberanilohydroxamic acid (SAHA), romidepsin, and panobinostat have been shown to induce HIV expression in these resting cells. Recently, it has been demonstrated that the low levels of viral gene expression induced by a candidate HDACi may be insufficient to cause the death of infected cells by viral cytopathic effects, necessitating their elimination by immune effectors, such as cytotoxic T-lymphocytes (CTL). Here, we study the impact of three HDACis in clinical development on T-cell effector functions. We report two modes of HDACi-induced functional impairment: i) the rapid suppression of cytokine production from viable T-cells induced by all three HDACis ii) the selective death of activated T-cells occurring at later time-points following transient exposures to romidepsin or, to a lesser extent, panobinostat. As a net result of these factors, HDACis impaired CTL-mediated IFN-γ production, as well as the elimination of HIV-infected or peptide-pulsed target cells, both in liquid culture and in collagen matrices. Romidepsin exerted greater inhibition of antiviral function than SAHA or panobinostat over the dose ranges tested. These data suggest that treatment with HDACis to mobilize the latent reservoir could have unintended negative impacts on the effector functions of CTL. This could influence the effectiveness of HDACi-based eradication strategies, by impairing elimination of infected cells, and is a critical consideration for trials where therapeutic interruptions are being contemplated, given the importance of CTL in containing rebound viremia.


Asunto(s)
Citotoxicidad Inmunológica/efectos de los fármacos , Depsipéptidos/farmacología , Infecciones por VIH/inmunología , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Indoles/farmacología , Linfocitos T Citotóxicos/inmunología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Panobinostat , Linfocitos T Citotóxicos/efectos de los fármacos , Replicación Viral/efectos de los fármacos
15.
J Immunol ; 192(2): 782-91, 2014 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-24337741

RESUMEN

CD8(+) CTLs are adept at killing virally infected cells and cancer cells and releasing cytokines (e.g., IFN-γ) to aid this response. However, during cancer and chronic viral infections, such as with HIV, this CTL response is progressively impaired due to a process called T cell exhaustion. Previous work has shown that the glycoprotein T cell Ig and mucin domain-containing protein 3 (Tim-3) plays a functional role in establishing T cell exhaustion. Tim-3 is highly upregulated on virus and tumor Ag-specific CD8(+) T cells, and antagonizing Tim-3 helps restore function of CD8(+) T cells. However, very little is known of how Tim-3 signals in CTLs. In this study, we assessed the role of Tim-3 at the immunological synapse as well as its interaction with proximal TCR signaling molecules in primary human CD8(+) T cells. Tim-3 was found within CD8(+) T cell lipid rafts at the immunological synapse. Blocking Tim-3 resulted in a significantly greater number of stable synapses being formed between Tim-3(hi)CD8(+) T cells and target cells, suggesting that Tim-3 plays a functional role in synapse formation. Further, we confirmed that Tim-3 interacts with Lck, but not the phospho-active form of Lck. Finally, Tim-3 colocalizes with receptor phosphatases CD45 and CD148, an interaction that is enhanced in the presence of the Tim-3 ligand, galectin-9. Thus, Tim-3 interacts with multiple signaling molecules at the immunological synapse, and characterizing these interactions could aid in the development of therapeutics to restore Tim-3-mediated immune dysfunction.


Asunto(s)
Proteínas de la Membrana/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Sinapsis/metabolismo , Linfocitos T CD8-positivos/metabolismo , Galectinas/metabolismo , Infecciones por VIH/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A , Humanos , Antígenos Comunes de Leucocito/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Transducción de Señal/fisiología
16.
J Immunol ; 193(11): 5576-83, 2014 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-25348621

RESUMEN

The T cell Ig- and mucin domain-containing molecule-3 (Tim-3) negative immune checkpoint receptor demarcates functionally exhausted CD8(+) T cells arising from chronic stimulation in viral infections like HIV. Tim-3 blockade leads to improved antiviral CD8(+) T cell responses in vitro and, therefore, represents a novel intervention strategy to restore T cell function in vivo and protect from disease progression. However, the Tim-3 pathway in the physiologically relevant rhesus macaque SIV model of AIDS remains uncharacterized. We report that Tim-3(+)CD8(+) T cell frequencies are significantly increased in lymph nodes, but not in peripheral blood, in SIV-infected animals. Tim-3(+)PD-1(+)CD8(+) T cells are similarly increased during SIV infection and positively correlate with SIV plasma viremia. Tim-3 expression was found primarily on effector memory CD8(+) T cells in all tissues examined. Tim-3(+)CD8(+) T cells have lower Ki-67 content and minimal cytokine responses to SIV compared with Tim-3(-)CD8(+) T cells. During acute-phase SIV replication, Tim-3 expression peaked on SIV-specific CD8(+) T cells by 2 wk postinfection and then rapidly diminished, irrespective of mutational escape of cognate Ag, suggesting non-TCR-driven mechanisms for Tim-3 expression. Thus, rhesus Tim-3 in SIV infection partially mimics human Tim-3 in HIV infection and may serve as a novel model for targeted studies focused on rejuvenating HIV-specific CD8(+) T cell responses.


Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/inmunología , Linfocitos T CD8-positivos/inmunología , Inmunoterapia/métodos , Proteínas de la Membrana/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/fisiología , Síndrome de Inmunodeficiencia Adquirida/terapia , Secuencia de Aminoácidos , Animales , Linfocitos T CD8-positivos/virología , Proliferación Celular , Células Cultivadas , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Receptor 2 Celular del Virus de la Hepatitis A , Humanos , Memoria Inmunológica , Macaca mulatta , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Terapia Molecular Dirigida , Receptor de Muerte Celular Programada 1/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/terapia , Carga Viral , Replicación Viral
17.
PLoS Pathog ; 9(3): e1003241, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23555254

RESUMEN

To develop new approaches to control HIV-1 replication, we examined the capacity of recently described small molecular modulators of RNA splicing for their effects on viral RNA metabolism. Of the drugs tested, digoxin was found to induce a dramatic inhibition of HIV-1 structural protein synthesis, a response due, in part, to reduced accumulation of the corresponding viral mRNAs. In addition, digoxin altered viral RNA splice site use, resulting in loss of the essential viral factor Rev. Digoxin induced changes in activity of the CLK family of SR protein kinases and modification of several SR proteins, including SRp20 and Tra2ß, which could account for the effects observed. Consistent with this hypothesis, overexpression of SRp20 elicited changes in HIV-1 RNA processing similar to those observed with digoxin. Importantly, digoxin was also highly active against clinical strains of HIV-1 in vitro, validating this novel approach to treatment of this infection.


Asunto(s)
Antivirales/farmacología , Digoxina/farmacología , Inhibidores Enzimáticos/farmacología , VIH-1/efectos de los fármacos , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Antígenos CD4/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Regulación Viral de la Expresión Génica/efectos de los fármacos , Genes rev/efectos de los fármacos , VIH-1/genética , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Proteínas del Tejido Nervioso/metabolismo , Empalme del ARN/efectos de los fármacos , ARN Viral/efectos de los fármacos , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Empalme Serina-Arginina , Proteínas Virales
18.
J Virol ; 87(24): 13307-20, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24089548

RESUMEN

Type 1 long-interspersed nuclear elements (L1s) are autonomous retrotransposable elements that retain the potential for activity in the human genome but are suppressed by host factors. Retrotransposition of L1s into chromosomal DNA can lead to genomic instability, whereas reverse transcription of L1 in the cytosol has the potential to activate innate immune sensors. We hypothesized that HIV-1 infection would compromise cellular control of L1 elements, resulting in the induction of retrotransposition events. Here, we show that HIV-1 infection enhances L1 retrotransposition in Jurkat cells in a Vif- and Vpr-dependent manner. In primary CD4(+) cells, HIV-1 infection results in the accumulation of L1 DNA, at least the majority of which is extrachromosomal. These data expose an unrecognized interaction between HIV-1 and endogenous retrotransposable elements, which may have implications for the innate immune response to HIV-1 infection, as well as for HIV-1-induced genomic instability and cytopathicity.


Asunto(s)
ADN Viral/metabolismo , Retrovirus Endógenos/genética , Infecciones por VIH/virología , VIH-1/genética , Elementos de Nucleótido Esparcido Largo , Linfocitos T CD4-Positivos/virología , Línea Celular , ADN Viral/genética , Retrovirus Endógenos/metabolismo , Infecciones por VIH/genética , VIH-1/metabolismo , Humanos , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo
19.
J Virol ; 87(11): 6073-80, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23536679

RESUMEN

APOBEC3 proteins mediate potent antiretroviral activity by hypermutating the retroviral genome during reverse transcription. To counteract APOBEC3 and gain a replicative advantage, lentiviruses such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) have evolved the Vif protein, which targets APOBEC3 proteins for proteasomal degradation. However, the proteasome plays a critical role in the generation of T cell peptide epitopes. Whether Vif-mediated destruction of APOBEC3 proteins leads to the generation and presentation of APOBEC3-derived T cell epitopes on the surfaces of lentivirus-infected cells remains unknown. Here, using peptides derived from multiple Vif-sensitive APOBEC3 proteins, we identified APOBEC3-specific T cell responses in both HIV-1-infected patients and SIV-infected rhesus macaques. These results raise the possibility that these T cell responses may be part of the larger antiretroviral immune response.


Asunto(s)
Linfocitos T CD8-positivos/virología , Citidina Desaminasa/inmunología , Citosina Desaminasa/inmunología , Infecciones por VIH/enzimología , VIH-1/fisiología , Síndrome de Inmunodeficiencia Adquirida del Simio/enzimología , Virus de la Inmunodeficiencia de los Simios/fisiología , Desaminasa APOBEC-3G , Adulto , Animales , Linfocitos T CD8-positivos/inmunología , Citidina Desaminasa/genética , Citosina Desaminasa/genética , Femenino , Productos del Gen vif/genética , Productos del Gen vif/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/inmunología , Humanos , Macaca mulatta , Masculino , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología
20.
J Immunol ; 188(8): 4008-22, 2012 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-22427638

RESUMEN

Neisseria gonorrhoeae, the cause of the sexually transmitted infection gonorrhea, elicits low levels of specific Ig that decline rapidly after the bacteria are cleared. Reinfection with the same serovar can occur, and prior gonococcal infection does not alter the Ig response upon subsequent exposure, suggesting that protective immunity is not induced. The mucosal Ig response apparent during gonorrhea does not correlate with that observed systemically, leading to a suggestion that it is locally generated. In considering whether N. gonorrhoeae directly influences B cells, we observed that gonococcal infection prolonged viability of primary human B cells in vitro and elicited robust activation and vigorous proliferative responses in the absence of T cells. Furthermore, we observed the specific expansion of IgD(+)CD27(+) B cells in response to gonococcal infection. These cells are innate in function, conferring protection against diverse microbes by producing low-affinity, broadly reactive IgM without inducing classical immunologic memory. Although gonococcal infection of B cells produced small amounts of gonococcal-specific IgM, IgM specific for irrelevant Ags were also produced, suggesting a broad, polyspecific Ig response. The gonococci were effectively bound and engulfed by B cells. TLR9-inhibitory CpGs blocked B cell responses, indicating that intracellular bacterial degradation allows for innate immune detection within the phagolysosome. To our knowledge, this is the first report of a bacterial pathogen having specific affinity for the human IgM memory B cells, driving their potent activation and polyclonal Ig response. This unfocused T-independent response explains the localized Ig response that occurs, despite an absence of immunologic memory elicited during gonorrhea.


Asunto(s)
Anticuerpos Antibacterianos/inmunología , Linfocitos B/inmunología , Inmunidad Innata , Inmunoglobulina M/biosíntesis , Neisseria gonorrhoeae/inmunología , Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/inmunología , Linfocitos B/microbiología , Proliferación Celular , Células Cultivadas , Gonorrea/inmunología , Gonorrea/microbiología , Humanos , Inmunoglobulina D/biosíntesis , Inmunoglobulina D/inmunología , Inmunoglobulina M/inmunología , Memoria Inmunológica , Masculino , Oligonucleótidos/farmacología , Fagocitosis , Fagosomas/inmunología , Receptor Toll-Like 9/antagonistas & inhibidores , Receptor Toll-Like 9/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología
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