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1.
Blood ; 133(7): 697-709, 2019 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-30463995

RESUMEN

Chimeric antigen receptor T (CAR-T) cell therapy is a new pillar in cancer therapeutics; however, its application is limited by the associated toxicities. These include cytokine release syndrome (CRS) and neurotoxicity. Although the IL-6R antagonist tocilizumab is approved for treatment of CRS, there is no approved treatment of neurotoxicity associated with CD19-targeted CAR-T (CART19) cell therapy. Recent data suggest that monocytes and macrophages contribute to the development of CRS and neurotoxicity after CAR-T cell therapy. Therefore, we investigated neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential strategy to manage CART19 cell-associated toxicities. In this study, we show that GM-CSF neutralization with lenzilumab does not inhibit CART19 cell function in vitro or in vivo. Moreover, CART19 cell proliferation was enhanced and durable control of leukemic disease was maintained better in patient-derived xenografts after GM-CSF neutralization with lenzilumab. In a patient acute lymphoblastic leukemia xenograft model of CRS and neuroinflammation (NI), GM-CSF neutralization resulted in a reduction of myeloid and T cell infiltration in the central nervous system and a significant reduction in NI and prevention of CRS. Finally, we generated GM-CSF-deficient CART19 cells through CRISPR/Cas9 disruption of GM-CSF during CAR-T cell manufacturing. These GM-CSFk/o CAR-T cells maintained normal functions and had enhanced antitumor activity in vivo, as well as improved overall survival, compared with CART19 cells. Together, these studies illuminate a novel approach to abrogate NI and CRS through GM-CSF neutralization, which may potentially enhance CAR-T cell function. Phase 2 studies with lenzilumab in combination with CART19 cell therapy are planned.


Asunto(s)
Citocinas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Enfermedades del Sistema Inmune/terapia , Inflamación/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos de Linfocitos T/uso terapéutico , Receptores Quiméricos de Antígenos/inmunología , Animales , Anticuerpos Neutralizantes/farmacología , Proliferación Celular , Humanos , Enfermedades del Sistema Inmune/inmunología , Enfermedades del Sistema Inmune/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Síndrome , Trasplante Heterólogo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
2.
J Immunol ; 201(3): 1086-1096, 2018 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-29914885

RESUMEN

The bone marrow microenvironment harbors and protects leukemic cells from apoptosis-inducing agents via mechanisms that are incompletely understood. We previously showed SDF-1 (CXCL-12), a chemokine readily abundant within the bone marrow microenvironment, induces apoptosis in acute myeloid leukemia (AML) cells that express high levels of the SDF-1 receptor CXCR4. However, differentiating osteoblasts found within this niche protect cocultured AML cells from apoptosis. Additionally, this protection was abrogated upon treatment of the differentiating osteoblasts with histone deacetylase inhibitors (HDACi). In this study, we begin to characterize and target the molecular mechanisms that mediate this osteoblast protection. Quantitative RT-PCR revealed that HDACi treatment of differentiating osteoblasts (mouse MC3T3 osteoblast cell line) reduced expression of multiple genes required for osteoblast differentiation, including genes important for producing mineralized bone matrix. Interestingly, pretreating differentiating osteoblasts with cyclosporine A, a drug known to inhibit osteoblast differentiation, similarly impaired osteoblast-mediated protection of cocultured AML cells (KG1a and U937 human AML cell lines). Both HDACi and cyclosporine A reduced osteoblast expression of the key mineralization enzyme tissue-nonspecific alkaline phosphatase (TNAP; encoded by Alpl). Moreover, specifically reducing TNAP expression or activity in differentiating osteoblasts significantly impaired the ability of the osteoblasts to protect cocultured AML cells. Together, our results indicate that inhibiting osteoblast matrix mineralization by specifically targeting TNAP is sufficient to significantly impair osteoblast-mediated protection of AML cells. Therefore, designing combination therapies that additionally target the osteoblast-produced mineralized bone matrix may improve treatment of AML by reducing the protection of leukemic cells within the bone marrow microenvironment.


Asunto(s)
Fosfatasa Alcalina/metabolismo , Apoptosis/fisiología , Leucemia Mieloide Aguda/metabolismo , Osteoblastos/metabolismo , Células 3T3 , Animales , Apoptosis/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Microambiente Celular/efectos de los fármacos , Quimiocina CXCL12/metabolismo , Técnicas de Cocultivo/métodos , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Ratones , Receptores CXCR4/metabolismo , Células U937
3.
J Biol Chem ; 293(36): 14022-14039, 2018 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-30018141

RESUMEN

The immune system includes abundant examples of biologically-relevant cross-regulation of signaling pathways by the T cell antigen receptor (TCR) and the G protein-coupled chemokine receptor, CXCR4. TCR ligation induces transactivation of CXCR4 and TCR-CXCR4 complex formation, permitting the TCR to signal via CXCR4 to activate a phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein (PREX1)-dependent signaling pathway that drives robust cytokine secretion by T cells. To understand this receptor heterodimer and its regulation, we characterized the molecular mechanisms required for TCR-mediated TCR-CXCR4 complex formation. We found that the cytoplasmic C-terminal domain of CXCR4 and specifically phosphorylation of Ser-339 within this region were required for TCR-CXCR4 complex formation. Interestingly, siRNA-mediated depletion of G protein-coupled receptor kinase-2 (GRK2) or inhibition by the GRK2-specific inhibitor, paroxetine, inhibited TCR-induced phosphorylation of CXCR4-Ser-339 and TCR-CXCR4 complex formation. Either GRK2 siRNA or paroxetine treatment of human T cells significantly reduced T cell cytokine production. Upstream, TCR-activated tyrosine kinases caused inducible tyrosine phosphorylation of GRK2 and were required for the GRK2-dependent events of CXCR4-Ser-339 phosphorylation and TCR-CXCR4 complex formation. Downstream of TCR-CXCR4 complex formation, we found that GRK2 and phosphatidylinositol 3-kinase γ (PI3Kγ) were required for TCR-stimulated membrane recruitment of PREX1 and for stabilization of cytokine mRNAs and robust cytokine secretion. Together, our results identify a novel role for GRK2 as a target of TCR signaling that is responsible for TCR-induced transactivation of CXCR4 and TCR-CXCR4 complex formation that signals via PI3Kγ/PREX1 to mediate cytokine production. Therapeutic regulation of GRK2 or PI3Kγ may therefore be useful for limiting cytokines produced by T cell malignancies or autoimmune diseases.


Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Citocinas/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Receptores de Antígenos de Linfocitos T/fisiología , Receptores CXCR4/metabolismo , Sitios de Unión , Humanos , Fosforilación , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Activación Transcripcional
4.
Blood ; 130(8): 982-994, 2017 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-28694325

RESUMEN

As with many immunopathologically driven diseases, the malignant T cells of cutaneous T-cell lymphomas (CTCLs), such as Sézary syndrome, display aberrant cytokine secretion patterns that contribute to pathology and disease progression. Targeting this disordered release of cytokines is complicated by the changing cytokine milieu that drives the phenotypic changes of CTCLs. Here, we characterize a novel signaling pathway that can be targeted to inhibit the secretion of cytokines by modulating either CXCR4 or CXCR4-mediated signaling. We demonstrate that upon ligation of the T-cell antigen receptor (TCR), the TCR associates with and transactivates CXCR4 via phosphorylation of S339-CXCR4 in order to activate a PREX1-Rac1-signaling pathway that stabilizes interleukin-2(IL-2), IL-4, and IL-10 messenger RNA (mRNA) transcripts. Pharmacologic inhibition of either TCR-CXCR4 complex formation or PREX1-Rac1 signaling in primary human T cells decreased mRNA stability and inhibited secretion of IL-2, IL-4, and IL-10. Applying this knowledge to Sézary syndrome, we demonstrate that targeting various aspects of this signaling pathway blocks both TCR-dependent and TCR-independent cytokine secretion from a Sézary syndrome-derived cell line and patient isolates. Together, these results identify multiple aspects of a novel TCR-CXCR4-signaling pathway that could be targeted to inhibit the aberrant cytokine secretion that drives the immunopathogenesis of Sézary syndrome and other immunopathological diseases.


Asunto(s)
Citocinas/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Linfoma Cutáneo de Células T/metabolismo , Estabilidad del ARN , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores CXCR4/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo , Bencilaminas , Ciclamas , Citocinas/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Humanos , Células Jurkat , Subgrupos Linfocitarios/efectos de los fármacos , Subgrupos Linfocitarios/metabolismo , Linfoma Cutáneo de Células T/patología , Modelos Biológicos , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Síndrome de Sézary/patología , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética
5.
J Biol Chem ; 290(49): 29478-92, 2015 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-26491017

RESUMEN

Disrupting the protective signals provided by the bone marrow microenvironment will be critical for more effective combination drug therapies for acute myeloid leukemia (AML). Cells of the osteoblast lineage that reside in the endosteal niche have been implicated in promoting survival of AML cells. Here, we investigated how to prevent this protective interaction. We previously showed that SDF-1, a chemokine abundant in the bone marrow, induces apoptosis of AML cells, unless the leukemic cells receive protective signals provided by differentiating osteoblasts (8, 10). We now identify a novel signaling pathway in differentiating osteoblasts that can be manipulated to disrupt the osteoblast-mediated protection of AML cells. Treating differentiating osteoblasts with histone deacetylase inhibitors (HDACi) abrogated their ability to protect co-cultured AML cells from SDF-1-induced apoptosis. HDACi prominently up-regulated expression of the Nherf1 scaffold protein, which played a major role in preventing osteoblast-mediated protection of AML cells. Protein phosphatase-1α (PP1α) was identified as a novel Nherf1 interacting protein that acts as the downstream mediator of this response by promoting nuclear localization of the TAZ transcriptional modulator. Moreover, independent activation of either PP1α or TAZ was sufficient to prevent osteoblast-mediated protection of AML cells even in the absence of HDACi. Together, these results indicate that HDACi target the AML microenvironment by enhancing activation of the Nherf1-PP1α-TAZ pathway in osteoblasts. Selective drug targeting of this osteoblast signaling pathway may improve treatments of AML by rendering leukemic cells in the bone marrow more susceptible to apoptosis.


Asunto(s)
Inhibidores de Histona Desacetilasas/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mieloide Aguda/metabolismo , Fosfoproteínas/metabolismo , Proteína Fosfatasa 1/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Microambiente Tumoral , Células 3T3 , Animales , Apoptosis , Médula Ósea/metabolismo , Diferenciación Celular , Núcleo Celular/metabolismo , Quimiocina CXCL12/metabolismo , Técnicas de Cocultivo , Humanos , Ratones , Osteoblastos/citología , Osteoblastos/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores CXCR4/metabolismo , Transducción de Señal , Fracciones Subcelulares/metabolismo , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ
6.
Mol Pharmacol ; 85(4): 542-52, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24452472

RESUMEN

CXC chemokine receptor 4 (CXCR4) is a G protein-coupled receptor (GPCR) located on the cell surface that signals upon binding the chemokine stromal derived factor-1 (SDF-1; also called CXCL 12). CXCR4 promotes neuroblastoma proliferation and chemotaxis. CXCR4 expression negatively correlates with prognosis and drives neuroblastoma growth and metastasis in mouse models. All functions of CXCR4 require its expression on the cell surface, yet the molecular mechanisms that regulate CXCR4 cell-surface levels in neuroblastoma are poorly understood. We characterized CXCR4 cell-surface regulation in the related SH-SY5Y and SK-N-SH human neuroblastoma cell lines. SDF-1 treatment caused rapid down-modulation of CXCR4 in SH-SY5Y cells. Pharmacologic activation of protein kinase C similarly reduced CXCR4, but via a distinct mechanism. Analysis of CXCR4 mutants delineated two CXCR4 regions required for SDF-1 treatment to decrease cell-surface CXCR4 in neuroblastoma cells: the isoleucine-leucine motif at residues 328 and 329 and residues 343-352. In contrast, and unlike CXCR4 regulation in other cell types, serines 324, 325, 338, and 339 were not required. Arrestin proteins can bind and regulate GPCR cell-surface expression, often functioning together with kinases such as G protein-coupled receptor kinase 2 (GRK2). Using SK-N-SH cells which are naturally deficient in ß-arrestin1, we showed that ß-arrestin1 is required for the CXCR4 343-352 region to modulate CXCR4 cell-surface expression following treatment with SDF-1. Moreover, GRK2 overexpression enhanced CXCR4 internalization, via a mechanism requiring both ß-arrestin1 expression and the 343-352 region. Together, these results characterize CXCR4 structural domains and ß-arrestin1 as critical regulators of CXCR4 cell-surface expression in neuroblastoma. ß-Arrestin1 levels may therefore influence the CXCR4-driven metastasis of neuroblastoma as well as prognosis.


Asunto(s)
Arrestinas/metabolismo , Quimiocina CXCL12/metabolismo , Neuroblastoma/metabolismo , Receptores CXCR4/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Quimiocina CXCL12/farmacología , Endocitosis , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Metástasis de la Neoplasia , Toxina del Pertussis/farmacología , Fosforilación , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Fosfolipasas de Tipo C/metabolismo , beta-Arrestinas
7.
J Biol Chem ; 288(32): 22899-914, 2013 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-23798675

RESUMEN

The CXCR4 chemokine receptor promotes survival of many different cell types. Here, we describe a previously unsuspected role for CXCR4 as a potent inducer of apoptosis in acute myeloid leukemia (AML) cell lines and a subset of clinical AML samples. We show that SDF-1, the sole ligand for CXCR4, induces the expected migration and ERK activation in the KG1a AML cell line transiently overexpressing CXCR4, but ERK activation did not lead to survival. Instead, SDF-1 treatment led via a CXCR4-dependent mechanism to apoptosis, as evidenced by increased annexin V staining, condensation of chromatin, and cleavage of both procaspase-3 and PARP. This SDF-1-induced death pathway was partially inhibited by hypoxia, which is often found in the bone marrow of AML patients. SDF-1-induced apoptosis was inhibited by dominant negative procaspase-9 but not by inhibition of caspase-8 activation, implicating the intrinsic apoptotic pathway. Further analysis showed that this pathway was activated by multiple mechanisms, including up-regulation of Bak at the level of mRNA and protein, stabilization of the Bak activator Noxa, and down-regulation of antiapoptotic Bcl-XL. Furthermore, adjusting expression levels of Bak, Bcl-XL, or Noxa individually altered the level of apoptosis in AML cells, suggesting that the combined modulation of these family members by SDF-1 coordinates their interplay to produce apoptosis. Thus, rather than mediating survival, SDF-1 may be a means to induce apoptosis of CXCR4-expressing AML cells directly in the SDF-1-rich bone marrow microenvironment if the survival cues of the bone marrow are disrupted.


Asunto(s)
Apoptosis , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Receptores CXCR4/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/biosíntesis , Proteína bcl-X/biosíntesis , Anexina A5/genética , Anexina A5/metabolismo , Supervivencia Celular/genética , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Regulación hacia Abajo/genética , Femenino , Células HEK293 , Humanos , Células Jurkat , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Masculino , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores CXCR4/genética , Células U937 , Regulación hacia Arriba/genética , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína bcl-X/genética
8.
J Cell Biochem ; 115(6): 1128-37, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24851270

RESUMEN

The bone marrow provides a protective environment for acute myeloid leukemia (AML) cells that often allows leukemic stem cells to survive standard chemotherapeutic regimens. Targeting these leukemic stem cells within the bone marrow is critical for preventing relapse. We recently demonstrated that SDF-1, a chemokine abundant in the bone marrow, induces apoptosis in AML cell lines and in patient samples expressing high levels of its receptor, CXCR4. Here we show that a subset of osteoblast lineage cells within the bone marrow can protect AML cells from undergoing apoptosis in response to the SDF-1 naturally present in that location. In co-culture systems, osteoblasts at various stages of differentiation protected AML cell lines and patient isolates from SDF-1-induced apoptosis. The differentiation of the osteoblast cell lines, MC3T3 and W-20-17, mediated this protection via a cell contact-independent mechanism. In contrast, bone marrow-derived mesenchymal cells, the precursors of osteoblasts, induced apoptosis in AML cells via a CXCR4-dependent mechanism and failed to protect AML cells from exogenously added SDF-1. These results indicate that osteoblasts in the process of differentiation potently inhibit the SDF-1-driven apoptotic pathway of CXCR4-expressing AML cells residing in the bone marrow. Drugs targeting this protective mechanism could potentially provide a new approach to treating AML by enhancing the SDF-1-induced apoptosis of AML cells residing within the bone marrow microenvironment.


Asunto(s)
Apoptosis/efectos de los fármacos , Quimiocina CXCL12/farmacología , Leucemia Mieloide/patología , Osteoblastos/citología , Enfermedad Aguda , Fosfatasa Alcalina/genética , Animales , Diferenciación Celular/genética , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , Citometría de Flujo , Expresión Génica , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , Osteoblastos/metabolismo , Osteocalcina/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células U937
9.
J Immunol ; 187(6): 3177-85, 2011 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-21856938

RESUMEN

RasGRP1, a Ras guanine-nucleotide exchange factor, critically mediates T cell development and function and controls immunodeficiency and autoimmunity. In this study, we describe a unique mechanism of mobilization and activation of RasGRP1 in response to SDF-1, a chemokine that signals via the G protein-coupled receptor CXCR4. Depletion of RasGRP1 impaired SDF-1-stimulated human T cell migration, expression of the activation marker CD69, and activation of the ERK MAPK pathway, indicating that RasGRP1 mediates SDF-1 functions. SDF-1 treatment caused RasGRP1 to localize to the plasma membrane to activate K-Ras and to the Golgi to activate N-Ras. These events were required for cellular migration and for ERK activation that mediates downstream transcriptional events in response to SDF-1. SDF-1-dependent localization of RasGRP1 did not require its diacylglycerol-binding domain, even though diacyglycerol was previously shown to mediate localization of RasGRP1 in response to Ag stimulation. This domain was, however, required for activity of RasGRP1 after its localization. Intriguingly, SDF-1 treatment of T cells induced the formation of a novel molecular signaling complex containing RasGRP1, Gαi2, and ZAP-70. Moreover, SDF-1-mediated signaling by both Gi proteins and ZAP-70 was required for RasGRP1 mobilization. In addition, RasGRP1 mobilization and activation in response to SDF-1 was dependent on TCR expression, suggesting that CXCR4 heterodimerizes with the TCR to couple to ZAP-70 and mobilize RasGRP1. These results increase understanding of the molecular mechanisms that mediate SDF-1 effects on T cells and reveal a novel mechanism of RasGRP1 regulation. Other G protein-coupled receptors may similarly contribute to regulation of RasGRP1.


Asunto(s)
Quimiocina CXCL12/inmunología , Proteínas de Unión al ADN/inmunología , Subunidad alfa de la Proteína de Unión al GTP Gi2/inmunología , Factores de Intercambio de Guanina Nucleótido/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Proteína Tirosina Quinasa ZAP-70/inmunología , Western Blotting , Membrana Celular/metabolismo , Quimiocina CXCL12/metabolismo , Quimiotaxis de Leucocito/inmunología , Proteínas de Unión al ADN/metabolismo , Activación Enzimática/inmunología , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Inmunoprecipitación , Activación de Linfocitos/inmunología , Transporte de Proteínas/inmunología , Linfocitos T/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismo
10.
J Immunol ; 186(2): 951-8, 2011 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-21148034

RESUMEN

CXCR4, like other G protein-coupled receptors, signals via heterotrimeric guanine nucleotide-binding proteins (G proteins) to regulate gene transcription, migration, development, growth, and transformation. We describe a formerly uncharacterized function of a G protein: a role in receptor trafficking. We previously showed that CXCR4 and the TCR physically associate and form a heterodimer upon stromal cell-derived factor-1 or CXCL12 (SDF-1) stimulation in human T cells to prolong ERK activation and, thereby, lead to gene upregulation and cytokine secretion. The CXCR4-TCR heterodimers occur on the cell surface and in an intracellular compartment in response to SDF-1. Neither the intracellular compartment to which the CXCR4-TCR heterodimers localize nor the mechanism for localization has been elucidated. In this article, we characterize molecular mechanisms required for postendocytic trafficking of CXCR4. Upon SDF-1 stimulation, CXCR4 localizes to Rab11(+) vesicles, a recycling compartment near the microtubule organizing center and Golgi apparatus. This trafficking requires the CXCR4 C-terminal tail domain but not the CXCR4 ubiquitination sites. The TCR also constitutively localizes to this Rab11(+) compartment. Trafficking of CXCR4 into the Rab11(+), TCR-containing endosomes requires actin polymerization. Furthermore, inhibiting Rho activation or depleting Gα13 prevented trafficking of CXCR4 into the Rab11(+) endosomes without hindering the ability of CXCR4 to endocytose. These results indicated that, upon SDF-1 treatment, Gα13 and Rho mediate the actin polymerization necessary for trafficking CXCR4 into the Rab11(+), recycling endosomal compartment, which also contains constitutively recycling TCR and, thus, CXCR4-TCR heterodimers. To our knowledge, this is the first report of Gα13 as a mediator of receptor trafficking.


Asunto(s)
Quimiocina CXCL12/fisiología , Vesículas Citoplasmáticas/metabolismo , Endosomas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/fisiología , Receptores CXCR4/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rho/fisiología , Vesículas Citoplasmáticas/inmunología , Endocitosis/inmunología , Endosomas/enzimología , Endosomas/inmunología , Humanos , Células Jurkat , Estructura Terciaria de Proteína , Transporte de Proteínas/inmunología , Receptores CXCR4/química , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Ubiquitinación/inmunología , Proteínas de Unión al GTP rab/biosíntesis
11.
J Immunol ; 187(3): 1440-7, 2011 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-21705626

RESUMEN

The CXCR4 chemokine receptor is a G protein-coupled receptor that signals in T lymphocytes by forming a heterodimer with the TCR. CXCR4 and TCR functions are consequently highly cross regulated, affecting T cell immune activation, cytokine secretion, and T cell migration. The CXCR4-TCR heterodimer stimulates T cell migration and activation of the ERK MAPK and downstream AP-1-dependent cytokine transcription in response to stromal cell-derived factor-1 (SDF-1), the sole chemokine ligand of CXCR4. These responses require Gi-type G proteins as well as TCR ITAM domains and the ZAP70 tyrosine kinase, thus indicating that the CXCR4-TCR heterodimer signals to integrate G protein-coupled receptor-associated and TCR-associated signaling molecules in response to SDF-1. Yet, the phospholipase C (PLC) isozymes responsible for coupling the CXCR4-TCR heterodimer to distinct downstream cellular responses are incompletely characterized. In this study, we demonstrate that PLC activity is required for SDF-1 to induce ERK activation, migration, and CXCR4 endocytosis in human T cells. SDF-1 signaling via the CXCR4-TCR heterodimer uses PLC-ß3 to activate the Ras-ERK pathway and increase intracellular calcium ion concentrations, whereas PLC-γ1 is dispensable for these outcomes. In contrast, PLC-γ1, but not PLC-ß3, is required for SDF-1-mediated migration via a mechanism independent of LAT. These results increase understanding of the signaling mechanisms employed by the CXCR4-TCR heterodimer, characterize new roles for PLC-ß3 and PLC-γ1 in T cells, and suggest that multiple PLCs may also be activated downstream of other chemokine receptors to distinctly regulate migration versus other signaling functions.


Asunto(s)
Quimiocina CXCL12/fisiología , Fosfolipasa C beta/fisiología , Fosfolipasa C gamma/fisiología , Multimerización de Proteína/inmunología , Receptores de Antígenos de Linfocitos T/fisiología , Receptores CXCR4/fisiología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Señalización del Calcio/inmunología , Movimiento Celular/inmunología , Endocitosis/inmunología , Humanos , Líquido Intracelular/enzimología , Líquido Intracelular/inmunología , Isoenzimas/fisiología , Células Jurkat , Sistema de Señalización de MAP Quinasas/inmunología , Receptores CXCR4/metabolismo , Subgrupos de Linfocitos T/enzimología
12.
J Immunol ; 187(1): 316-24, 2011 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-21613617

RESUMEN

CD4 Th cells are critical to the development of coordinated immune responses to infections and tumors. Th cells are activated through interactions of the TCR with MHC class II complexed with peptide. T cell activation is dependent on the density of MHC peptide complexes as well as the duration of interaction of the TCR with APCs. In this study, we sought to determine whether MHC class II peptides could be modified with amino acid sequences that facilitated uptake and presentation with the goal of improving Th cell activation in vitro and in vivo. A model epitope derived from the murine folate receptor α, a self- and tumor Ag, was modified at its carboxyl terminus with the invariant chain-derived Ii-Key peptide and at its N terminus with a peptide that enhances uptake of Ag by APC. Modification of a peptide resulted in enhanced generation of high-avidity murine folate receptor α T cells that persisted in vivo and homed to sites of Ag deposition. The nesting approach was epitope and species independent and specifically excluded expansion of CD4 regulatory T cells. The resulting Th cells were therapeutic, enhanced in vivo helper activity and had an increased ability to resist tolerizing immune microenvironments. In addition to improved immunoadjuvants, this epitope modification strategy may be useful for enhancing ex vivo and in vivo generation of Th cells for preventing and treating diseases.


Asunto(s)
Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Epítopos de Linfocito T/inmunología , Receptor 1 de Folato/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Secuencia de Aminoácidos , Animales , Adhesión Celular/inmunología , Línea Celular Tumoral , Movimiento Celular/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Epítopos de Linfocito T/uso terapéutico , Femenino , Receptor 1 de Folato/uso terapéutico , Antígenos de Histocompatibilidad Clase II/uso terapéutico , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Neoplasias Ováricas/terapia , Péptidos/inmunología , Péptidos/uso terapéutico , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Colaboradores-Inductores/patología
13.
Artículo en Inglés | MEDLINE | ID: mdl-38370136

RESUMEN

Tactics to increase the number of underrepresented (UR) students in biomedical research PhD training programs have not yet translated to UR faculty numbers that reflect the diversity of the United States. Continued interventions are required to build skills beyond those that result in placement into a PhD program. We hypothesize that successful interventions must build skills that give UR students foundations for confident self-efficacy in leadership. We seek interventions that allow UR students to envision themselves as successful faculty. We posit that development of such skills is difficult in the classroom or laboratory alone. Therefore, novel interventions are required. As part of the NIH-funded Post-baccalaureate Research Education Program (PREP) and Initiative for Maximizing Student Development (IMSD) at the Mayo Clinic Graduate School of Biomedical Sciences, we designed and implemented a unique intervention to support development of student leadership skills: a biannual student-organized and student-led national research conference titled "Scientific Innovation Through Diverse Perspectives" (SITDP). This initiative is based on the concept that students who actively live out realistic roles as scientific leaders will be encouraged to persist to scientific leadership as faculty. Here we describe the motivation for, design of, and outcomes from, the first three pilot conferences of this series. We further discuss approaches needed to rigorously evaluate the effectiveness of such interventions in the future.

14.
Mayo Clin Proc Innov Qual Outcomes ; 4(2): 203-210, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32280931

RESUMEN

OBJECTIVE: To evaluate the impact of a novel interdisciplinary graduate-level course in chimeric antigenic receptor-T cell therapy on students' knowledge and interests in translational science. MATERIALS/PARTICIPANTS AND METHODS: The course ran November 12 to 16, 2018. Students were surveyed before and after the course. The survey included questions regarding background, self-perceived knowledge/confidence in skills, and interests/predicted behaviors. Students were assigned to work in collaborative interdisciplinary teams to develop a research proposal. RESULTS: A total of 25 students taking the course for graduate-level credit were surveyed. Of these, all 25 (100%) completed the surveys. Students came from variable backgrounds and were at different stages of graduate training. After completion of the course, there was a statistically significant increase in self-perceived knowledge of immunotherapy (mean score of 3.6 postcourse vs 2.6 precourse, on a 5-point Likert scale; P<.001), knowledge of the bench to clinic translational process (3.7 postcourse vs 3.0 precourse; P<.001), confidence in critical reading skills (4.3 postcourse vs 4.0 precourse; P=.008), confidence in immunotherapy-focused grant writing skills (3.6 postcourse vs 2.8 precourse; P<.001), and interest in working in interdisciplinary teams (4.8 postcourse vs 4.6 precourse; P=.02). CONCLUSION: The structure of this innovative and comprehensive course serves as a platform for educational courses in interdisciplinary translational research and helps trainees build knowledge and interest in the fields of chimeric antigenic receptor-T cells, regenerative sciences, and immunotherapy.

15.
Oncotarget ; 8(55): 94569-94579, 2017 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-29212250

RESUMEN

The bone marrow microenvironment protects acute myeloid leukemia (AML) cells during chemotherapy and is a major factor in relapse. Here, we examined which type(s) of bone marrow cells are responsible for the relapse of AML following treatment with cytarabine (Ara-C), and we identified a means to inhibit this protection. To determine the protective cell type(s), AML cells were treated with Ara-C, and AML cell survival in the presence or absence of osteoblast lineage cells was assessed. Cultured AML cells and patient bone marrow isolates were each significantly protected from Ara-C-induced apoptosis by co-culture with differentiating osteoblasts. Moreover, pretreating differentiating osteoblasts with the histone deacetylase inhibitors (HDACi) vorinostat and panobinostat abrogated the ability of the differentiating osteoblasts to protect AML cells. Together, our results indicate that differentiating osteoblasts have the potential to promote residual AML in the bone marrow following standard chemotherapy and act via a mechanism requiring HDACi-sensitive gene expression. Using HDACi to target the leukemic microenvironment in combination with Ara-C could potentially improve treatment of AML. Moreover, other strategies for manipulating bone marrow osteoblasts may also help eradicate AML cells and reduce relapse.

16.
Cancer Res ; 76(2): 239-50, 2016 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-26567141

RESUMEN

The PD-1:PD-L1 immune signaling axis mediates suppression of T-cell-dependent tumor immunity. PD-1 expression was recently found to be upregulated on tumor-infiltrating murine (CD11c(+)CD11b(+)CD8(-)CD209a(+)) and human (CD1c(+)CD19(-)) myeloid dendritic cells (TIDC), an innate immune cell type also implicated in immune escape. However, there is little knowledge concerning how PD-1 regulates innate immune cells. In this study, we examined the role of PD-1 in TIDCs derived from mice bearing ovarian tumors. Similar to lymphocytes, TIDC expression of PD-1 was associated with expression of the adapter protein SHP-2, which signals to NF-κB; however, in contrast to its role in lymphocytes, we found that expression of PD-1 in TIDC tonically paralyzed NF-κB activation. Further mechanistic investigations showed that PD-1 blocked NF-κB-dependent cytokine release in a SHP-2-dependent manner. Conversely, inhibition of NF-κB-mediated antigen presentation by PD-1 occurred independently of SHP-2. Collectively, our findings revealed that PD-1 acts in a distinct manner in innate immune cells compared with adaptive immune cells, prompting further investigations of the signaling pathways controlled by this central mediator of immune escape in cancer.


Asunto(s)
Células Dendríticas/inmunología , FN-kappa B/metabolismo , Neoplasias Ováricas/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Animales , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias Ováricas/patología , Transducción de Señal
17.
J Cell Biol ; 210(2): 257-72, 2015 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-26195666

RESUMEN

IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a cytoskeleton-interacting scaffold protein. CXCR4 is a chemokine receptor that binds stromal cell-derived factor-1 (SDF-1; also known as CXCL12). Both IQGAP1 and CXCR4 are overexpressed in cancer cell types, yet it was unclear whether these molecules functionally interact. Here, we show that depleting IQGAP1 in Jurkat T leukemic cells reduced CXCR4 expression, disrupted trafficking of endocytosed CXCR4 via EEA-1(+) endosomes, and decreased efficiency of CXCR4 recycling. SDF-1-induced cell migration and activation of extracellular signal-regulated kinases 1 and 2 (ERK) MAPK were strongly inhibited, even when forced overexpression restored CXCR4 levels. Similar results were seen in KMBC and HEK293 cells. Exploring the mechanism, we found that SDF-1 treatment induced IQGAP1 binding to α-tubulin and localization to CXCR4-containing endosomes and that CXCR4-containing EEA-1(+) endosomes were abnormally located distal from the microtubule (MT)-organizing center (MTOC) in IQGAP1-deficient cells. Thus, IQGAP1 critically mediates CXCR4 cell surface expression and signaling, evidently by regulating EEA-1(+) endosome interactions with MTs during CXCR4 trafficking and recycling. IQGAP1 may similarly promote CXCR4 functions in other cancer cell types.


Asunto(s)
Endosomas/metabolismo , Receptores CXCR4/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Activadoras de ras GTPasa/fisiología , Movimiento Celular , Quimiocina CXCL12/metabolismo , Endocitosis , Células HEK293 , Humanos , Células Jurkat , Sistema de Señalización de MAP Quinasas , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Transporte de Proteínas , Receptores Opioides delta/metabolismo
18.
Mayo Clin Proc ; 79(3): 318-25, 2004 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15008605

RESUMEN

OBJECTIVE: To assess the clinical relevance of chemokine receptor expression on the progression of B-cell chronic lymphocytic leukemia (B-CLL). PATIENTS AND METHODS: Peripheral blood mononuclear cells from 45 patients with B-CLL were purified and compared with lymph node samples collected from 17 of these patients. Also compared were B cells obtained from peripheral blood samples from 5 healthy controls and B cells from reactive lymph nodes from 3 otherwise healthy persons. The patients were treated at the Mayo Clinic in Rochester, Minn, between January 15,1991, and February 7, 2003. Mononuclear cells were stained by a 2-color (fluorescein isothiocyanate/phycoerythrin) flow cytometric assay using antibodies to the chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR2, CCR4, CCR5, CCR6, and CCR7) and also to CD19. RESULTS: Of the 45 patients in this study, 20 had Rai stage 0 disease, 12 had stage I disease, 3 had stage II disease, 2 had stage III disease, and 8 had stage IV disease. The mean fluorescent intensity (MFI) of the chemokine receptor expression on B-CLL cells was compared with normal controls and was not significantly different, except for an increase in the median expression of CXCR3 (P = .003) and CCR7 (P = .001) on B-CLL cells. We also found a significant increase in the expression of CXCR4 and CCR7 in B-CLL cells from patients with stage IV compared with stage 0 disease (P = .001 and P = .02, respectively). Furthermore, circulating B-CLL cells showed significantly higher expression of CXCR4 and CCR7 when compared with B lymphocytes in lymph nodes (P = .003 and P < .001, respectively). CONCLUSION: The expression of CXCR4 and CCR7 on B-CLL cells correlates with Rai stage. Also, these chemokine receptors may be down-regulated once malignant B cells enter the lymph nodes. To our knowledge, this is the first published report that shows the strong association of Rai stage with CXCR4 and CCR7 expression levels in B-CLL cells.


Asunto(s)
Leucemia Linfocítica Crónica de Células B/metabolismo , Receptores de Quimiocina/metabolismo , ADP-Ribosil Ciclasa/análisis , ADP-Ribosil Ciclasa 1 , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/análisis , Linfocitos B/metabolismo , Biomarcadores de Tumor/metabolismo , Estudios de Casos y Controles , Progresión de la Enfermedad , Regulación hacia Abajo , Genes de Inmunoglobulinas/genética , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Enfermedades Linfáticas/metabolismo , Recuento de Linfocitos , Linfocitosis/metabolismo , Glicoproteínas de Membrana , Persona de Mediana Edad , Mutación , Estadificación de Neoplasias
19.
Int J Gastrointest Cancer ; 31(1-3): 23-9, 2002.
Artículo en Inglés | MEDLINE | ID: mdl-12622412

RESUMEN

The chemokines are a family of peptide hormones that regulate cellular adhesion, migration, proliferation, and survival. Accumulating evidence indicates critical regulatory roles for chemokines during the development of hyperplasias and metastatic tumors. Chemokines promote tumor metastasis, growth, survival, and angiogenesis. In addition, by regulating immunity, chemokines critically regulate anti-tumor immune responses and chronic inflammation such as that associated with various neoplasias. Since chemokine receptors are G-protein coupled receptors that are ideal drug targets, these discoveries presage the development of new and potent anti-cancer drugs that target chemokine receptors. Here, I review the chemokine system and chemokine regulation of neoplasias, with a special emphasis on pancreatic cancer.


Asunto(s)
Quimiocinas/farmacología , Proteínas de Unión al GTP/farmacología , Metástasis de la Neoplasia/inmunología , Metástasis de la Neoplasia/fisiopatología , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/fisiopatología , Apoptosis/fisiología , División Celular/fisiología , Transformación Celular Neoplásica , Humanos , Inflamación , Neovascularización Patológica/fisiopatología , Transducción de Señal
20.
PLoS One ; 9(1): e85090, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24454796

RESUMEN

Actin depolymerizing factor-homology (ADF-H) family proteins regulate actin filament dynamics at multiple cellular locations. Herein, we have investigated the function of the ADF-H family member coactosin-like 1 (COTL1) in the regulation of actin dynamics at the T cell immune synapse (IS). We initially identified COTL1 in a genetic screen to identify novel regulators of T cell activation, and subsequently found that it associates with F-actin and localizes at the IS in response to TCR+CD28 stimulation. Live cell microscopy showed that depletion of COTL1 protein impaired T cell spreading in response to TCR ligation and abrogated lamellipodial protrusion at the T cell - B cell contact site, producing only a band of F-actin. Significantly, re-expression of wild type COTL1, but not a mutant deficient in F-actin binding could rescue these defects. In addition, COTL1 depletion reduced T cell migration. In vitro studies showed that COTL1 and cofilin compete with each other for binding to F-actin, and COTL1 protects F-actin from cofilin-mediated depolymerization. While depletion of cofilin enhanced F-actin assembly and lamellipodial protrusion at the IS, concurrent depletion of both COTL1 and cofilin restored lamellipodia formation. Taken together, our results suggest that COTL1 regulates lamellipodia dynamics in part by protecting F-actin from cofilin-mediated disassembly.


Asunto(s)
Cofilina 1/antagonistas & inhibidores , Sinapsis Inmunológicas/metabolismo , Proteínas de Microfilamentos/metabolismo , Seudópodos/metabolismo , Actinas/metabolismo , Antígenos CD28/metabolismo , Movimiento Celular/efectos de los fármacos , Quimiocinas/farmacología , Cofilina 1/metabolismo , Prueba de Complementación Genética , Humanos , Sinapsis Inmunológicas/efectos de los fármacos , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Seudópodos/efectos de los fármacos , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología
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