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1.
Cell ; 185(16): 2918-2935.e29, 2022 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-35803260

RESUMEN

Neoadjuvant immune checkpoint blockade has shown promising clinical activity. Here, we characterized early kinetics in tumor-infiltrating and circulating immune cells in oral cancer patients treated with neoadjuvant anti-PD-1 or anti-PD-1/CTLA-4 in a clinical trial (NCT02919683). Tumor-infiltrating CD8 T cells that clonally expanded during immunotherapy expressed elevated tissue-resident memory and cytotoxicity programs, which were already active prior to therapy, supporting the capacity for rapid response. Systematic target discovery revealed that treatment-expanded tumor T cell clones in responding patients recognized several self-antigens, including the cancer-specific antigen MAGEA1. Treatment also induced a systemic immune response characterized by expansion of activated T cells enriched for tumor-infiltrating T cell clonotypes, including both pre-existing and emergent clonotypes undetectable prior to therapy. The frequency of activated blood CD8 T cells, notably pre-treatment PD-1-positive KLRG1-negative T cells, was strongly associated with intra-tumoral pathological response. These results demonstrate how neoadjuvant checkpoint blockade induces local and systemic tumor immunity.


Asunto(s)
Neoplasias , Receptor de Muerte Celular Programada 1 , Linfocitos T CD8-positivos , Humanos , Inmunoterapia , Linfocitos Infiltrantes de Tumor , Terapia Neoadyuvante , Neoplasias/terapia , Microambiente Tumoral
2.
Proc Natl Acad Sci U S A ; 117(1): 292-299, 2020 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-31879340

RESUMEN

We describe a Kappa-on-Heavy (KoH) mouse that produces a class of highly diverse, fully human, antibody-like agents. This mouse was made by replacing the germline variable sequences of both the Ig heavy-chain (IgH) and Ig kappa (IgK) loci with the human IgK germline variable sequences, producing antibody-like molecules with an antigen binding site made up of 2 kappa variable domains. These molecules, named KoH bodies, structurally mimic naturally existing Bence-Jones light-chain dimers in their variable domains and remain wild-type in their antibody constant domains. Unlike artificially diversified, nonimmunoglobulin alternative scaffolds (e.g., DARPins), KoH bodies consist of a configuration of normal Ig scaffolds that undergo natural diversification in B cells. Monoclonal KoH bodies have properties similar to those of conventional antibodies but exhibit an enhanced ability to bind small molecules such as the endogenous cardiotonic steroid marinobufagenin (MBG) and nicotine. A comparison of crystal structures of MBG bound to a KoH Fab versus a conventional Fab showed that the KoH body has a much deeper binding pocket, allowing MBG to be held 4 Å further down into the combining site between the 2 variable domains.


Asunto(s)
Anticuerpos/química , Anticuerpos/inmunología , Antígenos/inmunología , Cadenas Pesadas de Inmunoglobulina/química , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/química , Animales , Anticuerpos/genética , Anticuerpos/uso terapéutico , Secuencia de Bases , Sitios de Unión de Anticuerpos/genética , Bufanólidos , Ingeniería Genética , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Ratones , Modelos Moleculares , Nicotina , Conformación Proteica
3.
Nature ; 517(7534): 381-5, 2015 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-25561180

RESUMEN

Despite antiretroviral therapy (ART), human immunodeficiency virus (HIV)-1 persists in a stable latent reservoir, primarily in resting memory CD4(+) T cells. This reservoir presents a major barrier to the cure of HIV-1 infection. To purge the reservoir, pharmacological reactivation of latent HIV-1 has been proposed and tested both in vitro and in vivo. A key remaining question is whether virus-specific immune mechanisms, including cytotoxic T lymphocytes (CTLs), can clear infected cells in ART-treated patients after latency is reversed. Here we show that there is a striking all or none pattern for CTL escape mutations in HIV-1 Gag epitopes. Unless ART is started early, the vast majority (>98%) of latent viruses carry CTL escape mutations that render infected cells insensitive to CTLs directed at common epitopes. To solve this problem, we identified CTLs that could recognize epitopes from latent HIV-1 that were unmutated in every chronically infected patient tested. Upon stimulation, these CTLs eliminated target cells infected with autologous virus derived from the latent reservoir, both in vitro and in patient-derived humanized mice. The predominance of CTL-resistant viruses in the latent reservoir poses a major challenge to viral eradication. Our results demonstrate that chronically infected patients retain a broad-spectrum viral-specific CTL response and that appropriate boosting of this response may be required for the elimination of the latent reservoir.


Asunto(s)
Genes Dominantes/genética , Genes Virales/genética , VIH-1/genética , VIH-1/inmunología , Mutación/genética , Linfocitos T Citotóxicos/inmunología , Latencia del Virus/inmunología , Enfermedad Aguda/terapia , Animales , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Enfermedad Crónica/tratamiento farmacológico , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Infecciones por VIH/sangre , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/crecimiento & desarrollo , Humanos , Masculino , Ratones , ARN Viral/sangre , Carga Viral/efectos de los fármacos , Latencia del Virus/genética , Replicación Viral/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunología
4.
Proc Natl Acad Sci U S A ; 114(45): E9626-E9634, 2017 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-29078283

RESUMEN

Immunodeficient mice reconstituted with a human immune system represent a promising tool for translational research as they may allow modeling and therapy of human diseases in vivo. However, insufficient development and function of human natural killer (NK) cells and T cell subsets limit the applicability of humanized mice for studying cancer biology and therapy. Here, we describe a human interleukin 15 (IL15) and human signal regulatory protein alpha (SIRPA) knock-in mouse on a Rag2-/- Il2rg-/- background (SRG-15). Transplantation of human hematopoietic stem and progenitor cells into SRG-15 mice dramatically improved the development and functional maturation of circulating and tissue-resident human NK and CD8+ T cells and promoted the development of tissue-resident innate lymphoid cell (ILC) subsets. Profiling of human NK cell subsets by mass cytometry revealed a highly similar expression pattern of killer inhibitory receptors and other candidate molecules in NK cell subpopulations between SRG-15 mice and humans. In contrast to nonobese diabetic severe combined immunodeficient Il2rg-/- (NSG) mice, human NK cells in SRG-15 mice did not require preactivation but infiltrated a Burkitt's lymphoma xenograft and efficiently inhibited tumor growth following treatment with the therapeutic antibody rituximab. Our humanized mouse model may thus be useful for preclinical testing of novel human NK cell-targeted and combinatory cancer immunotherapies and for studying how they elicit human antitumor immune responses in vivo.


Asunto(s)
Células Asesinas Naturales/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Humanos , Inmunidad Innata/inmunología , Subunidad gamma Común de Receptores de Interleucina/inmunología , Interleucina-15/inmunología , Linfocitos/inmunología , Ratones , Ratones SCID , Receptores Inmunológicos/inmunología , Rituximab/inmunología
5.
Blood ; 129(8): 959-969, 2017 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-28077418

RESUMEN

Humanized mice are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, the existing models cannot support robust adaptive immune responses, especially the generation of class-switched, antigen-specific antibody responses. Here we describe a new mouse strain, in which human interleukin 6 (IL-6) gene encoding the cytokine that is important for B- and T-cell differentiation was knocked into its respective mouse locus. The provision of human IL-6 not only enhanced thymopoiesis and periphery T-cell engraftment, but also significantly increased class switched memory B cells and serum immunoglobulin G (IgG). In addition, immunization with ovalbumin (OVA) induced OVA-specific B cells only in human IL-6 knock-in mice. These OVA-specific antibodies displayed the highest frequency of somatic mutation, further suggesting that human IL-6 is important for efficient B-cell activation and selection. We conclude that human IL-6 knock-in mice represent a novel and improved model for human adaptive immunity without relying on complex surgery to transplant human fetal thymus and liver. These mice can therefore be used to exploit or evaluate immunization regimes that would be unethical or untenable in humans.


Asunto(s)
Inmunidad Adaptativa , Formación de Anticuerpos , Técnicas de Sustitución del Gen , Cambio de Clase de Inmunoglobulina , Interleucina-6/genética , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Pollos , Expresión Génica , Técnicas de Sustitución del Gen/métodos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Inmunización , Inmunoglobulina G/inmunología , Interleucina-6/inmunología , Ratones , Ovalbúmina/inmunología , Linfocitos T/citología , Linfocitos T/inmunología
6.
Commun Biol ; 6(1): 444, 2023 04 22.
Artículo en Inglés | MEDLINE | ID: mdl-37087494

RESUMEN

Immunodeficient mice reconstituted with a human immune system (HIS mice) give rise to human T cells, which make them an attractive system to study human immune responses to tumors. However, such HIS mice typically exhibit sub-optimal responses to immune challenges as well as fail to develop antigen-specific B or T cell memory. Here we report HIS mice mediate spontaneous regression of human B cell lymphoma Raji. Tumor regression was dependent on CD4+ and CD8+ T cell responses and resulted in T cell memory. The T cell memory elicited was mainly Raji-specific, however some level of cross-protection was also elicited to a related B cell lymphoma cell line Ramos. Single-cell RNAseq analysis indicated activation of CD8+ T cells in regressing Raji tumors as well as clonal expansion of specific T cell receptors (TCRs). Cloning of TCRs from Raji-infiltrating T cells into a Jurkat reporter cell line showed reactivity specific for Raji tumor cells. Overall, we report a platform for studying in vivo human T cell tumor immunity by highlighting spontaneous Raji tumor regression, clonal TCR expansion, and T cell memory in HIS mice.


Asunto(s)
Linfocitos T CD8-positivos , Linfoma de Células B , Humanos , Ratones , Animales , Receptores de Antígenos de Linfocitos T/metabolismo , Células Jurkat , Linfoma de Células B/metabolismo
7.
J Exp Med ; 203(4): 805-8, 2006 Apr 17.
Artículo en Inglés | MEDLINE | ID: mdl-16549599

RESUMEN

CD4+ T cells are classically thought to orchestrate adaptive immune responses. But recent studies demonstrate that they can also kill infected cells directly. A new paper shows that highly efficient processing of Epstein Barr virus (EBV) glycoproteins for presentation on MHC class II makes virus-transformed B cells susceptible to lysis by CD4+ T cells. Thus, antiviral vaccines should aim to stimulate both helper and cytolytic CD4+ T cells.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Citotoxicidad Inmunológica/inmunología , Animales , Humanos , Inmunidad Celular
8.
Cancer Immunol Res ; 10(10): 1190-1209, 2022 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-35895745

RESUMEN

Assessment of immune-cell subsets within the tumor immune microenvironment is a powerful approach to better understand cancer immunotherapy responses. However, the use of biopsies to assess the tumor immune microenvironment poses challenges, including the potential for sampling error, restricted sampling over time, and inaccessibility of some tissues/organs, as well as the fact that single biopsy analyses do not reflect discordance across multiple intrapatient tumor lesions. Immuno-positron emission tomography (PET) presents a promising translational imaging approach to address the limitations and assess changes in the tumor microenvironment. We have developed 89Zr-DFO-REGN5054, a fully human CD8A-specific antibody conjugate, to assess CD8+ tumor-infiltrating lymphocytes (TIL) pre- and posttherapy. We used multiple assays, including in vitro T-cell activation, proliferation, and cytokine production, and in vivo viral clearance and CD8 receptor occupancy, to demonstrate that REGN5054 has minimal impact on T-cell activity. Preclinical immuno-PET studies demonstrated that 89Zr-DFO-REGN5054 specifically detected CD8+ T cells in lymphoid tissues of CD8-genetically humanized immunocompetent mice (VelociT mice) and discerned therapy-induced changes in CD8+ TILs in two models of response to a CD20xCD3 T-cell activating bispecific antibody (REGN1979, odronextamab). Toxicology studies in cynomolgus monkeys showed no overt toxicity, and immuno-PET imaging in cynomolgus monkeys demonstrated dose-dependent clearance and specific targeting to lymphoid tissues. This work supports the clinical investigation of 89Zr-DFO-REGN5054 to monitor T-cell responses in patients undergoing cancer immunotherapy.


Asunto(s)
Anticuerpos Biespecíficos , Neoplasias , Animales , Linfocitos T CD8-positivos , Citocinas/uso terapéutico , Humanos , Linfocitos Infiltrantes de Tumor , Macaca fascicularis , Ratones , Tomografía de Emisión de Positrones/métodos , Radioisótopos , Microambiente Tumoral , Circonio
9.
Sci Immunol ; 6(66): eabj4026, 2021 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-34919442

RESUMEN

Despite the enormous promise of T cell therapies, the isolation and study of human T cell receptors (TCRs) of dedicated specificity remains a major challenge. To overcome this limitation, we generated mice with a genetically humanized system of T cell immunity. We used VelociGene technology to replace the murine TCRαß variable regions, along with regions encoding the extracellular domains of co-receptors CD4 and CD8, and major histocompatibility complex (MHC) class I and II, with corresponding human sequences. The resulting "VelociT" mice have normal myeloid and lymphoid immune cell populations, including thymic and peripheral αß T cell subsets comparable with wild-type mice. VelociT mice expressed a diverse TCR repertoire, mounted functional T cell responses to lymphocytic choriomeningitis virus infection, and could develop experimental autoimmune encephalomyelitis. Immunization of VelociT mice with human tumor-associated peptide antigens generated robust, antigen-specific responses and led to identification of a TCR against tumor antigen New York esophageal squamous cell carcinoma-1 with potent antitumor activity. These studies demonstrate that VelociT mice mount clinically relevant T cell responses to both MHC-I­ and MHC-II­restricted antigens, providing a powerful new model for analyzing T cell function in human disease. Moreover, VelociT mice are a new platform for de novo discovery of therapeutic human TCRs.


Asunto(s)
Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunología , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T alfa-beta/genética
10.
Blood ; 112(4): 1231-9, 2008 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-18519810

RESUMEN

Dendritic cells (DCs) express many endocytic receptors that deliver antigens for major histocompatibility class (MHC) I and II presentation to CD8(+) and CD4(+) T cells, respectively. Here, we show that targeting Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) to one of them, the human multilectin DEC-205 receptor, in the presence of the DC maturation stimulus poly(I:C), expanded EBNA1-specific CD4(+) and CD8(+) memory T cells, and these lymphocytes could control the outgrowth of autologous EBV-infected B cells in vitro. In addition, using a novel mouse model with reconstituted human immune system components, we demonstrated that vaccination with alphaDEC-205-EBNA1 antibodies primed EBNA1-specific IFN-gamma-secreting T cells and also induced anti-EBNA1 antibodies in a subset of immunized mice. Because EBNA1 is the one EBV antigen that is expressed in all proliferating cells infected with this virus, our data suggest that DEC-205 targeting should be explored as a vaccination approach against symptomatic primary EBV infection and against EBV-associated malignancies.


Asunto(s)
Antígenos CD/inmunología , Proliferación Celular , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Lectinas Tipo C/inmunología , Activación de Linfocitos , Receptores de Superficie Celular/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Antivirales , Linfocitos B/virología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Humanos , Memoria Inmunológica , Ratones , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor , Vacunación
11.
Oncoimmunology ; 9(1): 1758602, 2020 05 13.
Artículo en Inglés | MEDLINE | ID: mdl-32923116

RESUMEN

Clinical observations suggest that responses to cancer immunotherapy are correlated with intra-tumoral T cell receptor (TCR) clonality, tumor mutation burden (TMB) and host HLA genotype, highlighting the importance of host T cell recognition of tumor antigens. However, the dynamic interplay between T cell activation state and changes in TCR repertoire in driving the identification of potential immunodominant antigen(s) remains largely unexplored. Here, we performed single-cell RNA-sequencing on CD8+ tumor-infiltrating T cells (TILs) using the murine colorectal tumor model MC38 to identify unique TCR sequences and validate their tumor reactivity. We found that the majority of clonally expanded TILs are tumor-reactive and their TCR repertoire is unique amongst individual MC38 tumor-bearing mice. Our query identified that multiple expanded TCR clones recognized the retroviral epitope p15E as an immunodominant antigen. In addition, we found that the endogenous retroviral genome encoding for p15E is highly expressed in MC38 tumors, but not in normal tissues, due to epigenetic derepression. Further, we demonstrated that the p15E-specific TILs exhibit an activated phenotype and an increase in frequency upon treatment with anti-41BB and anti-PD-1 combination immunotherapy. Importantly, we showed that although p15E-specific TILs are not required to mount a primary anti-tumor response, they contributed to the development of strong immune memory. Overall our results revealed that endogenous retroviral antigens expressed by tumor cells may represent an important and underappreciated category of tumor antigens that could be readily targeted in the clinic.


Asunto(s)
Retrovirus Endógenos , Neoplasias , Animales , Inmunoterapia , Activación de Linfocitos , Ratones , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/genética
12.
Sci Transl Med ; 12(549)2020 06 24.
Artículo en Inglés | MEDLINE | ID: mdl-32581132

RESUMEN

Monoclonal antibodies that block the programmed cell death 1 (PD-1) checkpoint have revolutionized cancer immunotherapy. However, many major tumor types remain unresponsive to anti-PD-1 therapy, and even among responsive tumor types, most of the patients do not develop durable antitumor immunity. It has been shown that bispecific antibodies activate T cells by cross-linking the TCR/CD3 complex with a tumor-specific antigen (TSA). The class of TSAxCD3 bispecific antibodies have generated exciting results in early clinical trials. We have recently described another class of "costimulatory bispecifics" that cross-link a TSA to CD28 (TSAxCD28) and cooperate with TSAxCD3 bispecifics. Here, we demonstrate that these TSAxCD28 bispecifics (one specific for prostate cancer and the other for epithelial tumors) can also synergize with the broader anti-PD-1 approach and endow responsiveness-as well as long-term immune memory-against tumors that otherwise do not respond to anti-PD-1 alone. Unlike CD28 superagonists, which broadly activate T cells and induce cytokine storm, TSAxCD28 bispecifics display little or no toxicity when used alone or in combination with a PD-1 blocker in genetically humanized immunocompetent mouse models or in primates and thus may provide a well-tolerated and "off the shelf" combination approach with PD-1 immunotherapy that can markedly enhance antitumor efficacy.


Asunto(s)
Anticuerpos Biespecíficos , Neoplasias , Animales , Anticuerpos Biespecíficos/uso terapéutico , Antígenos CD28 , Humanos , Inmunoterapia , Ratones , Neoplasias/tratamiento farmacológico , Receptor de Muerte Celular Programada 1
13.
PLoS One ; 14(4): e0215132, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30998704

RESUMEN

BACKGROUND & AIMS: The early steps in the pathophysiology of celiac disease (CD) leading to loss of tolerance to gluten are poorly described. Our aim was to use RNA sequencing of duodenal biopsies in patients with active CD, CD in remission, and non-CD controls to gain insight into CD pathophysiology, identify additional genetic signatures linked to CD, and possibly uncover targets for future therapeutic agents. METHODS: We performed whole transcriptome shotgun sequencing of intestinal biopsies in subjects with active and remission CD and non-CD controls. We also performed functional pathway analysis of differentially expressed genes to identify statistically significant pathways that are up or down regulated in subjects with active CD compared to remission CD. RESULTS: We identified the upregulation of novel genes including IL12R, ITGAM and IGSF4 involved in the immune response machinery and cell adhesion process in the mucosa of subjects with active CD compared to those in remission. We identified a unique signature of genes, related to innate immunity, perturbed exclusively in CD irrespective of disease status. Finally, we highlight novel pathways of interest that may contribute to the early steps of CD pathogenesis and its comorbidities such as the spliceosome, pathways related to the innate immune response, and pathways related to autoimmunity. CONCLUSIONS: Our study confirmed previous findings based on GWAS and immunological studies pertinent to CD pathogenesis and describes novel genes and pathways that with further validation may be found to contribute to the early steps in the pathogenesis of CD, ongoing inflammation, and comorbidities associated with CD.


Asunto(s)
Biomarcadores/análisis , Enfermedad Celíaca/patología , Duodeno/metabolismo , Inflamación/genética , Mucosa Intestinal/metabolismo , Análisis de Secuencia de ARN/métodos , Estudios de Casos y Controles , Enfermedad Celíaca/genética , Enfermedad Celíaca/metabolismo , Humanos , Inflamación/complicaciones , Transducción de Señal , Transcriptoma
14.
Dis Model Mech ; 12(9)2019 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-31399482

RESUMEN

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer derived from the malignant transformation of T-cell progenitors. Outcomes remain poor for T-ALL patients who have either primary resistance to standard-of-care chemotherapy or disease relapse. Notably, there are currently no targeted therapies available in T-ALL. This lack of next-generation therapies highlights the need for relevant preclinical disease modeling to identify and validate new targets and treatment approaches. Here, we adapted a spontaneously arising, genetically heterogeneous, thymic transplantation-based murine model of T-ALL, recapitulating key histopathological and genetic features of the human disease, to the preclinical testing of targeted and immune-directed therapies. Genetic engineering of the murine Notch1 locus aligned the spectrum of Notch1 mutations in the mouse model to that of human T-ALL and confirmed aberrant, recombination-activating gene (RAG)-mediated 5' Notch1 recombination events as the preferred pathway in murine T-ALL development. Testing of Notch1-targeting therapeutic antibodies demonstrated T-ALL sensitivity to different classes of Notch1 blockers based on Notch1 mutational status. In contrast, genetic ablation of Notch3 did not impact T-ALL development. The T-ALL model was further applied to the testing of immunotherapeutic agents in fully immunocompetent, syngeneic mice. In line with recent clinical experience in T-cell malignancies, programmed cell death 1 (PD-1) blockade alone lacked anti-tumor activity against murine T-ALL tumors. Overall, the unique features of the spontaneous T-ALL model coupled with genetic manipulations and the application to therapeutic testing in immunocompetent backgrounds will be of great utility for the preclinical evaluation of novel therapies against T-ALL.


Asunto(s)
Inmunoterapia , Terapia Molecular Dirigida , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Receptores Notch/metabolismo , Transducción de Señal , Animales , Anticuerpos Monoclonales/metabolismo , Antígeno B7-H1/metabolismo , Modelos Animales de Enfermedad , Eliminación de Gen , Humanos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Oncogenes , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Timo/trasplante
15.
Sci Immunol ; 3(29)2018 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-30389797

RESUMEN

Most patients with cancer do not develop durable antitumor responses after programmed cell death protein 1 (PD-1) or programmed cell death ligand 1(PD-L1) checkpoint inhibition monotherapy because of an ephemeral reversal of T cell dysfunction and failure to promote long-lasting immunological T cell memory. Activating costimulatory pathways to induce stronger T cell activation may improve the efficacy of checkpoint inhibition and lead to durable antitumor responses. We performed single-cell RNA sequencing of more than 2000 tumor-infiltrating CD8+ T cells in mice receiving both PD-1 and GITR (glucocorticoid-induced tumor necrosis factor receptor-related protein) antibodies and found that this combination synergistically enhanced the effector function of expanded CD8+ T cells by restoring the balance of key homeostatic regulators CD226 and T cell immunoreceptor with Ig and ITIM domains (TIGIT), leading to a robust survival benefit. Combination therapy decreased CD8+ T cell dysfunction and induced a highly proliferative precursor effector memory T cell phenotype in a CD226-dependent manner. PD-1 inhibition rescued CD226 activity by preventing PD-1-Src homology region 2 (SHP2) dephosphophorylation of the CD226 intracellular domain, whereas GITR agonism decreased TIGIT expression. Unmasking the molecular pathways driving durable antitumor responses will be essential to the development of rational approaches to optimizing cancer immunotherapy.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Proteína Relacionada con TNFR Inducida por Glucocorticoide/inmunología , Memoria Inmunológica/inmunología , Inmunoterapia , Neoplasias/terapia , Receptor de Muerte Celular Programada 1/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/patología , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/inmunología , Fenotipo
16.
J Exp Med ; 206(6): 1423-34, 2009 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-19487422

RESUMEN

Many pathogens that cause human disease infect only humans. To identify the mechanisms of immune protection against these pathogens and also to evaluate promising vaccine candidates, a small animal model would be desirable. We demonstrate that primary T cell responses in mice with reconstituted human immune system components control infection with the oncogenic and persistent Epstein-Barr virus (EBV). These cytotoxic and interferon-gamma-producing T cell responses were human leukocyte antigen (HLA) restricted and specific for EBV-derived peptides. In HLA-A2 transgenic animals and similar to human EBV carriers, T cell responses against lytic EBV antigens dominated over recognition of latent EBV antigens. T cell depletion resulted in elevated viral loads and emergence of EBV-associated lymphoproliferative disease. Both loss of CD4(+) and CD8(+) T cells abolished immune control. Therefore, this mouse model recapitulates features of symptomatic primary EBV infection and generates T cell-mediated immune control that resists oncogenic transformation.


Asunto(s)
Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Sistema Inmunológico/inmunología , Neoplasias , Linfocitos T/inmunología , Animales , Modelos Animales de Enfermedad , Antígeno HLA-A2/inmunología , Humanos , Riñón/citología , Riñón/inmunología , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Neoplasias/inmunología , Neoplasias/virología , Bazo/citología , Bazo/inmunología , Linfocitos T/citología
17.
J Virol ; 79(2): 910-7, 2005 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15613319

RESUMEN

The p6 domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein mediates virion budding from infected cells via protein-protein contacts with the class E vacuolar protein sorting factors, Tsg101 and AIP1/ALIX. Interaction with Tsg101 is strengthened by covalent attachment of monovalent ubiquitin to HIV-1 p6. To identify additional host factors that bind to HIV-1 p6, a human cDNA library was screened in the yeast two-hybrid system. HIV-1 p6 was found to interact with small ubiquitin-like modifier 1 (SUMO-1) as well as the E2 SUMO-1 transfer enzyme, Ubc9. Interaction with p6 was also detected with Daxx, a cellular protein to which SUMO-1 is sometimes covalently attached. SUMO-1 was incorporated into HIV-1 virions where it was protected within the virion membrane from digestion by exogenous protease. Of the two lysine residues in p6, lysine 27 uniquely served as a site of covalent SUMO-1 attachment. As previously reported, though, HIV-1 bearing the p6-K27R mutation replicated just like the wild type. Overproduction of SUMO-1 in HIV-1 producer cells had no apparent effect on virion release or on virion protein or RNA content. Infectivity of the resulting virions, though, was decreased, with the defect occurring after membrane fusion, at the time of viral cDNA synthesis. HIV-1 bearing the p6-K27R mutation was insensitive to SUMO-1 overexpression, suggesting that covalent attachment of SUMO-1 to p6 is detrimental to HIV-1 replication.


Asunto(s)
Productos del Gen gag/metabolismo , VIH-1/fisiología , Proteína SUMO-1/fisiología , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Bases , Proteínas Portadoras/fisiología , Línea Celular , Proteínas Co-Represoras , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Chaperonas Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/fisiología , Relación Estructura-Actividad , Enzimas Ubiquitina-Conjugadoras/fisiología , Replicación Viral
18.
J Virol ; 79(9): 5557-67, 2005 May.
Artículo en Inglés | MEDLINE | ID: mdl-15827170

RESUMEN

Heat shock protein 70 (Hsp70) is incorporated within the membrane of primate lentiviral virions. Here we demonstrate that Hsp70 is also incorporated into oncoretroviral virions and that it remains associated with membrane-stripped human immunodeficiency virus type 1 (HIV-1) virion cores. To determine if Hsp70 promotes virion infectivity, we attempted to generate Hsp70-deficient virions with gag deletion mutations, Hsp70 transdominant mutants, or RNA interference, but these efforts were confounded, largely because they disrupt virion assembly. Given that polypeptide substrates are bound and released by Hsp70 in an ATP-hydrolytic reaction cycle, we supposed that incubation of HIV-1 virions with ATP would perturb Hsp70 interaction with substrates in the virion and thereby decrease infectivity. Treatment with ATP or ADP had no observable effect, but ATPgammaS and GTPgammaS, nucleotide triphosphate analogues resistant to Hsp70 hydrolysis, dramatically reduced the infectivity of HIV-1 and murine leukemia virus virions. ATPgammaS-treated virions were competent for fusion with susceptible target cells, but viral cDNA synthesis was inhibited to an extent that correlated with the magnitude of decrease in infectivity. Intravirion reverse transcription by HIV-1, simian immunodeficiency virus, or murine leukemia virus was also inhibited by ATPgammaS. The effects of ATPgammaS on HIV-1 reverse transcription appeared to be indirect, resulting from disruption of virion core morphology that was evident by transmission electron microscopy. Consistent with effects on capsid conformation, ATPgammaS-treated viruslike particles failed to saturate host antiviral restriction activity. Our observations support a model in which the catalytic activity of virion-associated Hsp70 is required to maintain structural integrity of the virion core.


Asunto(s)
Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , VIH-1/fisiología , Virus de la Leucemia Murina/química , Virión/fisiología , Animales , Línea Celular , Eliminación de Gen , Genes gag/genética , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Infecciones por VIH/virología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/química , VIH-1/patogenicidad , VIH-1/ultraestructura , Proteínas HSP70 de Choque Térmico/deficiencia , Proteínas HSP70 de Choque Térmico/genética , Humanos , Virus de la Leucemia Murina/genética , Ratas , Infecciones por Retroviridae/virología , Inhibidores de la Transcriptasa Inversa/farmacología , Infecciones Tumorales por Virus/virología , Virión/química , Virión/ultraestructura , Virulencia , Ensamble de Virus
19.
J Virol ; 76(9): 4666-70, 2002 May.
Artículo en Inglés | MEDLINE | ID: mdl-11932435

RESUMEN

To determine if any heat shock proteins are incorporated into human immunodeficiency virus type 1 (HIV-1) virions in a manner similar to that of the peptidyl-prolyl isomerase cyclophilin A, we probed purified virions with antibodies against heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70, Hsc70, and Hsp90. Of these proteins, Hsp60, Hsp70, and Hsc70 associated with virions purified based on either particle density or size and were shown to be incorporated within the virion membrane, where they were protected from digestion by exogenous protease. Virion incorporation of Hsp70 was also observed with HIV-2 and with simian immunodeficiency viruses SIV(MAC) and SIV(AGM), but it appears to be specific for primate lentiviruses, since Hsp70 was not detected in association with Moloney murine leukemia virus virions. Of the HIV-1 genes, gag was found to be sufficient for Hsp70 incorporation, though Hsp70 was roughly equimolar with pol-encoded proteins in virions.


Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Lentivirus/metabolismo , Primates , Virión/metabolismo , Animales , Línea Celular , VIH-1/metabolismo , VIH-2/metabolismo , Humanos , Virus de la Inmunodeficiencia de los Simios/metabolismo , Transfección
20.
J Virol ; 77(5): 3167-80, 2003 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-12584341

RESUMEN

Potent drugs such as cyclosporine have provided effective probes of signal transduction pathways and, as well, of human immunodeficiency virus type 1 (HIV-1) replication mechanisms. Recently, it was reported that As(2)O(3), a drug used to treat acute promyelocytic leukemia (PML), stimulates HIV-1 replication. We found that As(2)O(3) accelerates the kinetics of a spreading HIV-1 infection in human T cells and increases the number of cells bearing HIV-1 provirus after a single round of infection. The stimulatory effect occurred after membrane fusion and resulted in increased steady-state levels of newly synthesized viral cDNA. Stimulation was independent of HIV-1 env and most viral accessory genes, and As(2)O(3) had no detectable effects on viral expression postintegration or virion assembly. Murine leukemia virus (MLV) transduction was enhanced by As(2)O(3) to the same extent as HIV-1 transduction, but As(2)O(3) had no additional effect on Fv1 restriction. In contrast, As(2)O(3) largely overcame the specific block to N-tropic MLV reverse transcription posed by human Ref1. As(2)O(3) disrupts PML bodies, nuclear structures named for a major component, the PML protein. We observed no changes in PML bodies in response to HIV-1 infection. Experiments with PML-null target cells indicated that PML has no effect on HIV-1 infectivity and is dispensable for the stimulatory effect of As(2)O(3). As(2)O(3) caused cell death in uninfected cells at the same concentrations which stimulate HIV-1 replication. Among four additional apoptosis-inducing agents, a boost in HIV-1 infectivity was observed only with carbonyl cyanide m-chlorophenylhydrazone, a compound which, like As(2)O(3), disrupts the mitochondrial transmembrane potential. In summary, As(2)O(3) stimulates retroviral reverse transcription, perhaps via effects on mitochondria, and provides a useful tool for characterizing Ref1.


Asunto(s)
Arsenicales/farmacología , Liasas de Carbono-Oxígeno/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa , VIH-1/efectos de los fármacos , Óxidos/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Trióxido de Arsénico , Línea Celular , ADN Complementario/biosíntesis , VIH-1/genética , VIH-1/fisiología , Células HeLa , Humanos , Células Jurkat , Virus de la Leucemia Murina/efectos de los fármacos , Virus de la Leucemia Murina/genética , Virus de la Leucemia Murina/metabolismo , Fusión de Membrana , Ratones , Proteínas/metabolismo , Transducción Genética , Replicación Viral/efectos de los fármacos
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