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Over the past decade, US Food and Drug Administration (FDA)-approved immune checkpoint inhibitors that target programmed death-1 (PD-1) have demonstrated significant clinical benefit particularly in patients with PD-L1 expressing tumors. Toripalimab is a humanized anti-PD-1 antibody, approved by FDA for first-line treatment of nasopharyngeal carcinoma in combination with chemotherapy. In a post hoc analysis of phase 3 studies, toripalimab in combination with chemotherapy improved overall survival irrespective of PD-L1 status in nasopharyngeal carcinoma (JUPITER-02), advanced non-small cell lung cancer (CHOICE-01) and advanced esophageal squamous cell carcinoma (JUPITER-06). On further characterization, we determined that toripalimab is molecularly and functionally differentiated from pembrolizumab, an anti-PD-1 mAb approved previously for treating a wide spectrum of tumors. Toripalimab, which binds the FG loop of PD-1, has 12-fold higher binding affinity to PD-1 than pembrolizumab and promotes significantly more Th1- and myeloid-derived inflammatory cytokine responses in healthy human PBMCs in vitro. In an ex vivo system employing dissociated tumor cells from treatment naïve non-small cell lung cancer patients, toripalimab induced several unique genes in IFN-γ and immune cell pathways, showed different kinetics of activation and significantly enhanced IFN-γ signature. Additionally, binding of toripalimab to PD-1 induced lower levels of SHP1 and SHP2 recruitment, the negative regulators of T cell activation, in Jurkat T cells ectopically expressing PD-1. Taken together, these data demonstrate that toripalimab is a potent anti-PD-1 antibody with high affinity PD-1 binding, strong functional attributes and demonstrated clinical activity that encourage its continued clinical investigation in several types of cancer.
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Anticuerpos Monoclonales Humanizados , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Neoplasias Pulmonares , Neoplasias Nasofaríngeas , Humanos , Anticuerpos Monoclonales/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/patología , Antígeno B7-H1 , Receptor de Muerte Celular Programada 1 , Neoplasias Pulmonares/tratamiento farmacológico , Carcinoma Nasofaríngeo , Neoplasias Esofágicas/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Linfocitos T/patologíaRESUMEN
Natural killer (NK) cells are cytotoxic innate lymphocytes that are specialized to kill tumor cells. NK cells are responsive to the primary cytokine IL-2 in the tumor microenvironment (TME), to activate its effector functions against tumors. Despite their inherent ability to kill tumor cells, dysfunctional NK cells observed within advanced solid tumors are associated with poor patient survival. Hypoxia in the TME is a major contributor to immune evasion in solid tumors that could contribute to impaired NK cell function. HIF-1α is a nodal regulator of hypoxia in driving the adaptive cellular responses to changes in oxygen concentrations. Whether HIF-1α is expressed in hypoxic NK cells in the context of IL-2 and whether its expression regulates NK cell effector function are unclear. Here, we report that freshly isolated NK cells from human peripheral blood in hypoxia could not stabilize HIF-1α protein coincident with impaired anti-tumor cytotoxicity. However, ex vivo expansion of these cells restored HIF-1α levels in hypoxia to promote antitumor cytotoxic functions. Similarly, the human NK cell line NKL expressed HIF-1α upon IL-2 stimulation in hypoxia and exhibited improved anti-tumor cytotoxicity and IFN-γ secretion. We found that ex vivo expanded human NK cells and NKL cells required the concerted activation of PI3K/mTOR pathway initiated by IL-2 signaling in combination with hypoxia for HIF-1α stabilization. These findings highlight that HIF-1α stabilization in hypoxia maximizes NK cell effector function and raises the prospect of NK cells as ideal therapeutic candidates for solid tumors.
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Subunidad alfa del Factor 1 Inducible por Hipoxia , Interleucina-2 , Células Asesinas Naturales , Neoplasias , Hipoxia de la Célula , Línea Celular Tumoral , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Interleucina-2/metabolismo , Interleucina-2/farmacología , Células Asesinas Naturales/inmunología , Neoplasias/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Fibronectin (FN) is an extracellular matrix (ECM) glycoprotein that self-assembles into FN fibrils, forming a FN matrix contributing to the stiffness of the ECM. Stromal FN stiffness in cancer has been shown to impact epithelial functions such as migration, cancer metastasis, and epithelial-to-mesenchymal transition. The role of the FN matrix of epithelial cells in driving such processes remains less well understood and is the focus of this study. Hypoxia, defined by low oxygen tension (<5%) is one of the hallmarks of tumor microenvironments impacting fibril reorganization in stromal and epithelial cells. Here, using the MCF10 breast epithelial progression series of cell lines encompassing normal, preinvasive, and invasive states, we show that FN fibril formation decreases during hypoxia, coinciding with a decrease in migratory potential of these cells. Conversely, we find that FN fibril disruption during three-dimensional acinar growth of normal breast cells resulted in acinar luminal filling. Our data also demonstrates that the luminal filling upon fibril disruption in untransformed MCF10A cells results in a loss of apicobasal polarity, characteristic of pre-invasive and invasive breast cell lines MCF10AT and MCF10 DCIS.com. Overall this is the first study that relates fibril-mediated changes in epithelial cells as critical players in lumen clearing of breast acini and maintenance of the untransformed growth characteristic.
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Movimiento Celular/fisiología , Fibronectinas/metabolismo , Células Acinares/citología , Células Acinares/metabolismo , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Movimiento Celular/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Citometría de Flujo , Humanos , Immunoblotting , InmunohistoquímicaRESUMEN
The delivery of immunomodulators directly into the tumor potentially harnesses the existing antigen, tumor-specific infiltrating lymphocytes, and antigen presenting cells. This can confer specificity and generate a potent systemic anti-tumor immune response with lower doses and less toxicity compared to systemic administration, in effect an in situ vaccine. Here, we test this concept using the novel combination of immunomodulators anti-CTLA4, -CD137, and -OX40. The triple combination administered intratumorally at low doses to one tumor of a dual tumor mouse model had dramatic local and systemic anti-tumor efficacy in lymphoma (A20) and solid tumor (MC38) models, consistent with an abscopal effect. The minimal effective dose was 10 µg each. The effect was dependent on CD8 T-cells. Intratumoral administration resulted in superior local and distant tumor control compared to systemic routes, supporting the in situ vaccine concept. In a single tumor A20 model, injection close to the tDLN resulted in similar efficacy as intratumoral and significantly better than targeting a non-tDLN, supporting the role of the tDLN as a viable immunotherapy target in addition to the tumor itself. Distribution studies confirmed expected concentration of antibodies in tumor and tDLN, in keeping with the anti-tumor results. Overall intratumoral or peri-tDLN administration of the novel combination of anti-CTLA4, anti-CD137, and anti-OX40, all agents in the clinic or clinical trials, demonstrates potent systemic anti-tumor effects. This immunotherapeutic combination is promising for future clinical development via both these safe and highly efficacious routes of administration.
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Anticuerpos Monoclonales/uso terapéutico , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Neoplasias del Colon/terapia , Inmunoterapia/métodos , Linfoma/terapia , Ganglio Linfático Centinela/patología , Animales , Antígeno CTLA-4/inmunología , Neoplasias del Colon/inmunología , Modelos Animales de Enfermedad , Quimioterapia Combinada , Femenino , Humanos , Linfoma/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores OX40/inmunología , Inducción de Remisión , Ganglio Linfático Centinela/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunologíaRESUMEN
B cells internalize extracellular Ag into endosomes using the Ig component of the BCR. In endosomes, Ag-derived peptides are loaded onto MHC class II proteins. How these pathways intersect remains unclear. We find that HLA-DM (DM), a catalyst for MHC class II peptide loading, coprecipitates with Ig in lysates from human tonsillar B cells and B cell lines. The molecules in the Ig/DM complexes have mature glycans, and the complexes colocalize with endosomal markers in intact cells. A larger fraction of Ig precipitates with DM after BCR crosslinking, implying that complexes can form when DM meets endocytosed Ig. In vitro, in the endosomal pH range, soluble DM directly binds the Ig Fab domain and increases levels of free Ag released from immune complexes. Taken together, these results argue that DM and Ig intersect in the endocytic pathway of B cells with potential functional consequences.
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Linfocitos B/inmunología , Antígenos HLA-D/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Compartimento Celular/inmunología , Línea Celular Tumoral , Endosomas/inmunología , Humanos , Tonsila Palatina/citología , Tonsila Palatina/inmunologíaRESUMEN
OBJECTIVE: In the K/BxN mouse model of inflammatory arthritis, T cells carrying a transgenic T cell receptor initiate disease by helping B cells to produce arthritogenic anti-glucose-6-phosphate isomerase (anti-GPI) autoantibodies. We found that lethally- irradiated lymphocyte-deficient C57BL/6 (B6).g7 (I-A(g7) +) recombinase-activating gene-deficient (Rag(-/-)) mice reconstituted with K/BxN hematopoietic stem and progenitor cells exhibit arthritis by week 4. In contrast, healthy B6.g7 recipients of K/BxN hematopoietic stem and progenitor cells show only mild arthritis, with limited extent and duration. The objective of this study was to investigate the factors responsible for the attenuation of arthritis in B6.g7 recipients. METHODS: Antibody responses were measured by enzyme-linked immunosorbent assay. Fluorescence-activated cell sorting analyses were performed for testing chimerism, expression of markers of activation and suppression, tetramer binding, and intracellular cytokines in CD4+ T cells. Suppressive activity of CD4+ T cells was studied by adoptive transfer. RESULTS: Titers of anti-GPI antibodies in reconstituted B6.g7 mice were â¼60-fold lower than in reconstituted B6.g7 Rag(-/-) mice. Examination of chimerism in the reconstituted B6.g7 mice showed that B cells and myeloid cells in these mice were donor derived, but CD4+ T cells were primarily host derived and enriched for cells expressing the conventional regulatory markers CD25 and FoxP3. Notably, CD4+CD25-FoxP3- T cells expressed markers of suppressive function (CD73 and folate receptor 4), and delayed disease after adoptive transfer. Activation of donor-derived CD4+ T cells was reduced, and thymic deletion of these cells appeared increased. CONCLUSION: Despite myeloablation, host CD4+ T cells having a regulatory phenotype emerge in these mice and attenuate autoimmunity.
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Artritis/inmunología , Enfermedades Autoinmunes/inmunología , Linfocitos T CD4-Positivos/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Células Madre Hematopoyéticas/inmunología , 5'-Nucleotidasa/metabolismo , Traslado Adoptivo , Animales , Artritis/etiología , Artritis/patología , Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/patología , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/metabolismo , Glucosa-6-Fosfato Isomerasa/genética , Glucosa-6-Fosfato Isomerasa/inmunología , Células Madre Hematopoyéticas/patología , Proteínas de Homeodominio/genética , Memoria Inmunológica/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Receptores de Superficie Celular/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Timo/citología , Timo/inmunología , Irradiación Corporal TotalRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a fatal disease defined by a progressive decline in lung function due to scarring and accumulation of extracellular matrix (ECM) proteins. The SOCS (Suppressor Of Cytokine Signaling) domain is a 40 amino acid conserved domain known to form a functional ubiquitin ligase complex targeting the Von Hippel Lindau (VHL) protein for proteasomal degradation. Here we show that the SOCS conserved domain operates as a molecular tool, to disrupt collagen and fibronectin fibrils in the ECM associated with fibrotic lung myofibroblasts. Our results demonstrate that fibroblasts differentiated using TGFß, followed by transduction with the SOCS domain, exhibit significantly reduced levels of the contractile myofibroblast-marker, α-SMA. Furthermore, in support of its role to retard differentiation, we find that lung fibroblasts expressing the SOCS domain present with significantly reduced levels of α-SMA and fibrillar fibronectin after differentiation with TGFß. We show that adenoviral delivery of the SOCS domain in the fibrotic phase of experimental lung fibrosis in mice, significantly reduces collagen accumulation in disease lungs. These data underscore a novel function for the SOCS domain and its potential in ameliorating pathologic matrix deposition in lung fibroblasts and experimental lung fibrosis.
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Several MHC class II alleles linked with autoimmune diseases form unusually low-stability complexes with class II-associated invariant chain peptides (CLIP), leading us to hypothesize that this is an important feature contributing to autoimmune pathogenesis. We recently demonstrated a novel post-endoplasmic reticulum (ER) chaperoning role of the CLIP peptides for the murine class II allele I-E(d). In the current study, we tested the generality of this CLIP chaperone function using a series of invariant chain (Ii) mutants designed to have varying CLIP affinity for I-A(g7). In cells expressing these Ii CLIP mutants, I-A(g7) abundance, turnover and antigen presentation are all subject to regulation by CLIP affinity, similar to I-E(d). However, I-A(g7) undergoes much greater quantitative changes than observed for I-E(d). In addition, we find that Ii with a CLIP region optimized for I-A(g7) binding may be preferentially assembled with I-A(g7) even in the presence of higher levels of wild-type Ii. This finding indicates that, although other regions of Ii interact with class II, CLIP binding to the groove is likely to be a dominant event in assembly of nascent class II molecules with Ii in the ER.
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Antígenos de Diferenciación de Linfocitos B/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Chaperonas Moleculares/inmunología , Procesamiento Proteico-Postraduccional/inmunología , Animales , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Linfocitos B/inmunología , Línea Celular Tumoral , Citometría de Flujo , Antígenos de Histocompatibilidad Clase II/genética , Immunoblotting , Ratones , Ratones Endogámicos BALB C , MutaciónRESUMEN
Monocytes are crucial regulators of inflammation, and are characterized by three distinct subsets in humans, of which classical and non-classical are the most abundant. Different subsets carry out different functions and have been previously associated with multiple inflammatory conditions. Dissecting the contribution of different monocyte subsets to disease is currently limited by samples and cohorts, often resulting in underpowered studies and poor reproducibility. Publicly available transcriptome profiles provide an alternative source of data characterized by high statistical power and real-world heterogeneity. However, most transcriptome datasets profile bulk blood or tissue samples, requiring the use of in silico approaches to quantify changes in cell levels. Here, we integrated 853 publicly available microarray expression profiles of sorted human monocyte subsets from 45 independent studies to identify robust and parsimonious gene expression signatures, consisting of 10 genes specific to each subset. These signatures maintain their accuracy regardless of disease state in an independent cohort profiled by RNA-sequencing and are specific to their respective subset when compared to other immune cells from both myeloid and lymphoid lineages profiled across 6160 transcriptome profiles. Consequently, we show that these signatures can be used to quantify changes in monocyte subsets levels in expression profiles from patients in clinical trials. Finally, we show that proteins encoded by our signature genes can be used in cytometry-based assays to specifically sort monocyte subsets. Our results demonstrate the robustness, versatility, and utility of our computational approach and provide a framework for the discovery of new cellular markers.
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Biomarcadores , Monocitos/metabolismo , Transcriptoma , Plasticidad de la Célula , Biología Computacional , Susceptibilidad a Enfermedades , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Homeostasis , Humanos , Inmunofenotipificación , Monocitos/inmunología , Transducción de SeñalRESUMEN
Two types of single-walled carbon nanotubes (SWCNTs), HiPco- and carboxyl-SWCNT, are evaluated as drug carriers for the traditional anti-inflammatory drug methotrexate (MTX) and a small interfering RNA (siRNA) targeting NOTCH1 gene. The nanotubes are solubilized by PEGylation and covalently loaded with MTX. The coupling efficiency (CE%) of MTX is 77-79% for HiPco-SWCNT and 71-83% for carboxyl-SWCNT. siRNA is noncovalently attached to the nanotubes with efficiency of 90-97% for HiPco-SWCNT and 87-98% for carboxyl-SWCNT. Through whole body imaging in the second near-infrared window (NIR-II window, 1000-1700 nm), SWCNTs were found to be selectively accumulated in inflamed joints in a serum transfer mouse model. We further investigated the interactions of the siRNA/MTX loaded nanotubes with human blood and mice bone marrow cells. In human blood, both types of unloaded SWCNTs were associated with B cells, monocytes and neutrophils. Interestingly, loading with MTX suppressed SWCNTs targeting specificity to immune cells, especially B cells; in contrast, loading siRNA alone enhanced the targeting specificity. Loading both MTX and siRNA to carboxyl-SWCNT enhanced targeting specificity to neutrophils and monocytes but not B cells. The targeting specificity of SWCNTs can potentially be adjusted by altering the ratio of MTX and siRNA loaded. The combined results show that carbon nanotubes have the potential for delivery of cargo drugs specifically to immune cells involved in rheumatoid arthritis.
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Serum transfer from arthritic K/BxN mice into naive animals results in arthritis. Mast cells have been shown to be essential since mice lacking these cell type do not develop arthritis upon serum injection. Mast cell function depends on the release of granules filled with mediators such as histamine. Mice deficient in histidine decarboxylase (HDC(-/-)) that do not produce histamine and mice deficient for histamine receptor 1 (H1R(-/-)) or histamine receptor 2 (H2R(-/-)) were injected with arthritogenic sera from the K/BxN mice, and the progression of arthritis was observed through the next 2 weeks. HDC(-/-) mice that are histamine free developed a milder form of arthritis in comparison with the wild-type controls. In both receptor-deficient mice as well as in wild-type controls, the onset and severity of clinical arthritis and ankle thickening occurred during day 1 to 3. These results indicate that histamine is required but not indispensable for the development of serum-induced arthritis and histamine receptors other than those studied here may be involved.
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Artritis Experimental/inmunología , Histamina/metabolismo , Histidina Descarboxilasa/metabolismo , Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H2/metabolismo , Animales , Artritis Experimental/genética , Artritis Experimental/patología , Histidina Descarboxilasa/genética , Mastocitos/inmunología , Ratones , Ratones Noqueados , Receptores Histamínicos H1/genética , Receptores Histamínicos H2/genéticaRESUMEN
Fibronectin (FN) is a core matrix protein that assembles to form a dynamic cellular scaffold, frequently perturbed during oncogenic transformation. Tumor hypoxia, characterized by low oxygen concentrations in the microenvironment of most solid tumors has been shown to accelerate FN assembly in fibroblasts and cancer-associated fibroblasts, cell types that produce abundant amounts of FN protein. Nevertheless, FN matrix regulation in epithelial cancer cells during hypoxia remains less well defined. In this study we investigate the assembly of the FN matrix during hypoxia in renal cancer epithelial cells, the cells of origin of renal cell carcinoma (RCC). We show that hypoxia (1% O2) specifically increases matrix disassembly and increases migratory propensity in renal cancer cells. However, HIFα stabilization using hypoxia mimetics, does not recapitulate the effect of hypoxia on FN matrix reorganization or cell migration. Using a combination of knockdown and inhibitor-based approaches, our work characterizes the signaling events that mediate these two disparate changes on the matrix and explores its functional significance on chemotactic cell migration. Our study systematically reexamines the role of hypoxia mimetics as experimental substitutes for hypoxia and provides new findings on HIFα stabilization and the FN matrix in the context of renal cancer.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Células Renales/metabolismo , Cobalto/farmacología , Fibronectinas/metabolismo , Hipoxia/metabolismo , Neoplasias Renales/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Humanos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/fisiologíaRESUMEN
The dominant cancer treatment modalities such as chemotherapy, radiotherapy, and even targeted kinase inhibitors and mAbs are limited by low efficacy, toxicity, and treatment-resistant tumor subclones. Oncolytic viral therapy offers a novel therapeutic strategy that has the potential to dramatically improve clinical outcomes. Reovirus, a double-stranded benign human RNA virus, is a leading candidate for therapeutic development and currently in phase III trials. Reovirus selectively targets transformed cells with activated Ras signaling pathways; Ras genes are some of the most frequently mutated oncogenes in human cancer and it is estimated that at least 30% of all human tumors exhibit aberrant Ras signaling. By targeting Ras-activated cells, reovirus can directly lyse cancer cells, disrupt tumor immunosuppressive mechanisms, reestablish multicellular immune surveillance, and generate robust antitumor responses. Reovirus therapy is currently being tested in combination with radiotherapy, chemotherapy, immunotherapy, and surgery. In this review, we discuss the current successes of these combinatorial therapeutic strategies and emphasize the importance of prioritizing combination oncolytic viral therapy as reovirus-based treatments progress in clinical development. Mol Cancer Ther; 15(5); 767-73. ©2016 AACR.
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Vectores Genéticos , Neoplasias/terapia , Viroterapia Oncolítica , Virus Oncolíticos , Reoviridae , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Terapia Combinada , Vectores Genéticos/genética , Humanos , Inmunoterapia , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Virus Oncolíticos/fisiología , Radioterapia/métodos , Reoviridae/fisiología , Transducción de Señal , Replicación ViralRESUMEN
Type I diabetes (T1D), mediated by autoreactive T cell destruction of insulin-producing islet beta cells, has been treated with bone marrow-derived hematopoietic stem cell (BM-HSC) transplantation. Older non-obese diabetic (NOD) mice recipients (3m, at disease-onset stage) receiving syngeneic BM-HSC progressed more rapidly to end-stage diabetes post-transplantation than younger recipients (4-6w, at disease-initiation stage). FACS analyses showed a higher percentage and absolute number of regulatory T cells (Treg) and lower proportion of proliferating T conventional cells (Tcon) in pancreatic lymph nodes from the resistant mice among the younger recipients compared to the rapid progressors among the older recipients. Treg distribution in spleen, mesenteric lymph nodes (MLN), blood and thymus between the two groups was similar. However, the percentage of thymic Tcon and the proliferation of Tcon in MLN and blood were lower in the young resistants. These results suggest recipient age and associated disease stage as a variable to consider in BM-HSC transplantation for treating T1D.
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Envejecimiento/inmunología , Diabetes Mellitus Tipo 1/inmunología , Trasplante de Células Madre Hematopoyéticas , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Envejecimiento/patología , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Biomarcadores/metabolismo , Glucemia/inmunología , Glucemia/metabolismo , Diabetes Mellitus Tipo 1/mortalidad , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/terapia , Modelos Animales de Enfermedad , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Inmunofenotipificación , Recuento de Linfocitos , Ratones , Ratones Endogámicos NOD , Análisis de Supervivencia , Subgrupos de Linfocitos T/patología , Linfocitos T Reguladores/patología , Irradiación Corporal TotalRESUMEN
The therapeutic efficacy of some anti-tumor monoclonal antibodies (mAbs) depends on the capacity of the mAb to recognize the tumor-associated antigen and induce cytotoxicity via a network of immune effector cells. This process of antibody-dependent cell-mediated cytotoxicity (ADCC) against tumor cells is triggered by the interaction of the fragment crystallizable (Fc) portion of the mAb with the Fc receptors on effector cells like natural killer cells, macrophages, γδ T cells, and dendritic cells. By augmenting ADCC, the antitumor activity of mAbs can be significantly increased. Currently, identifying and developing therapeutic agents that enhance ADCC is a growing area of research. Combining existing tumor-targeting mAbs and ADCC-promoting agents that stimulate effector cells will translate to greater clinical responses. In this review, we discuss strategies for enhancing ADCC and emphasize the potential of combination treatments that include US Food and Drug Administration-approved mAbs and immunostimulatory therapeutics.
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Gene therapy mediated by bone marrow-derived hematopoietic stem cells (BM-HSC) has been widely used in treating genetic deficiencies in both pre-clinical and clinical settings. Using mitotically inactive cell-targeting lentivirus with separate promoters for our gene of interest (the murine MHC class II (MHCII) chaperone, invariant chain (Ii)) and a GFP reporter, we monitored the expression and function of introduced Ii in various types of professional antigen presenting cells (B cells, macrophages and DC) from different organs (spleen, pancreatic lymph nodes (PLN), BM and blood). Ii and GFP were detected. Ii levels correlated with GFP levels only in macrophages and monocytes from spleen, monocytes from PLN and macrophage precursors from blood. By cell type, Ii levels in PLN cells were more similar to those in spleen cells than to those in blood or BM cells. Functionally, Ii expressed in PLN or spleen had more effect on MHCII abundance than Ii expressed in BM or blood. The results have implications for analysis of the outcomes of gene therapy when both therapeutic and reporter genes are introduced. The findings also have implications for understanding the development of immune molecule function.
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Células de la Médula Ósea/citología , Regulación de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Transgenes/genética , Animales , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/metabolismo , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/metabolismo , Células Cultivadas , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Hematopoyéticas/citología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Lentivirus/genética , Ganglios Linfáticos/metabolismo , Ratones , Regiones Promotoras Genéticas , Bazo/metabolismoRESUMEN
INTRODUCTION: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial hyperplasia and progressive cartilage and bone destruction that leads to a substantial loss of general functions and/or a decline in physical activities such as walking speed in humans. The K/BxN serum transfer arthritis in mice shares many immunological and pathological features with human RA. Very few studies are available in mice that investigate the changes in physical activity in relation to arthritis development. In this study we investigate the effect of arthritis on the locomotor activity of mice during K/BxN sera transfer arthritis. METHODS: Arthritis was induced in Balb/c mice by injecting intraperitoneally with 200ul of K/BxN sera; Balb/c mice injected with phosphate buffered saline (PBS) served as control. Progress of arthritis was estimated by daily measurements of joint thickness. Each mouse's locomotor activity (travel distance and travel time) was assessed every day for duration of 20 minute period using the SmartCage™ platform. Data were analyzed using the SmartCage™ analysis software (CageScore™). RESULTS: Arthritic Balb/c mice showed a reduction in distance covered and travel speed when compared to arthritis-free, control Balb/c mice. Maximum decline in locomotor activity was observed during the peak period of the disease and correlated to the increase in joint thickness in the arthritic mice. CONCLUSION: This report demonstrates that measuring locomotor activity of mice during progression of K/BxN sera-induced arthritis using the SmartCage™ platform offers a quantitative method to assess physical activity in mice during arthritis.
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HIV-derived lentiviral vectors have been used widely to transduce non-dividing cells, such as hematopoietic stem cells (HSCs), in the setting of gene therapy. In this study, we screened lentiviral vectors for their ability to drive expression of the murine MHC class II chaperone, invariant chain (Ii) and a GFP reporter. The vectors included T2A vector with T2A-separated Ii and GFP under the same MSCV promoter, dual-promoter vectors with separate promoters for Ii and GFP (called MSCV or EF1a according to the promoter driving Ii expression), and a vector with EF1a driving a fusion of Ii/GFP (called Fusion vector). T2A and MSCV induced the highest levels of Ii and GFP expression, respectively, after direct transfection of 293T cells. All vectors except the Fusion vector drove expression of functional Ii, based on the enhancement of MHC class II level, which is a known consequence of Ii expression. Comparing the vectors after they were packaged into lentiviruses and used to transduce 293T, we found that MSCV and EF1a vectors mediated higher Ii and GFP expression. In ckit(+) bone marrow (BM) cells, MSCV still induced the highest Ii and GFP expression, whereas EF1a induced only robust Ii expression. Regardless of the vector, both Ii and GFP levels were significantly reduced in BM cells compared to 293T cells. When in vivo expression was assessed in cells derived from MSCV-transduced BM-HSCs, up to 80% of myeloid cells were GFP(+), but no Ii expression was observed. In contrast, transplantation of EF1a-transduced BM-HSCs led to much higher in vivo Ii expression. Thus, among those compared, dual-promoter vector-based lentivirus with the EF1a promoter driving the gene of interest is optimal for murine BM-HSC transduction.
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Médula Ósea/metabolismo , Proteínas Fluorescentes Verdes/genética , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/metabolismo , Lentivirus/genética , Animales , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/metabolismo , Citometría de Flujo , Regulación de la Expresión Génica , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Ratones , Ratones Endogámicos NOD , Factor 1 de Elongación Peptídica/genética , Regiones Promotoras Genéticas/genética , Retroviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción GenéticaRESUMEN
INTRODUCTION: Matrix metalloproteinases (MMPs) are important in tissue remodelling. Here we investigate the role of collagenase-3 (MMP-13) in antibody-induced arthritis. METHODS: For this study we employed the K/BxN serum-induced arthritis model. Arthritis was induced in C57BL/6 wild type (WT) and MMP-13-deficient (MMP-13/) mice by intraperitoneal injection of 200 µl of K/BxN serum. Arthritis was assessed by measuring the ankle swelling. During the course of the experiments, mice were sacrificed every second day for histological examination of the ankle joints. Ankle sections were evaluated histologically for infiltration of inflammatory cells, pannus tissue formation and bone/cartilage destruction. Semi-quantitative PCR was used to determine MMP-13 expression levels in ankle joints of untreated and K/BxN serum-injected mice. RESULTS: This study shows that MMP-13 is a regulator of inflammation. We observed increased expression of MMP-13 in ankle joints of WT mice during K/BxN serum-induced arthritis and both K/BxN serum-treated WT and MMP-13/ mice developed progressive arthritis with a similar onset. However, MMP-13/ mice showed significantly reduced disease over the whole arthritic period. Ankle joints of WT mice showed severe joint destruction with extensive inflammation and erosion of cartilage and bone. In contrast, MMP-13/ mice displayed significantly decreased severity of arthritis (50% to 60%) as analyzed by clinical and histological scoring methods. CONCLUSIONS: MMP-13 deficiency acts to suppress the local inflammatory responses. Therefore, MMP-13 has a role in the pathogenesis of arthritis, suggesting MMP-13 is a potential therapeutic target.