Agradecemos al Dr. Shinya Yamanaka (Universidad de Kyoto) para proporcionar iPS-MEF-Ng-20D-17 línea celular, el Dr. Darío Vignali (Hospital St. Jude Childrens Research) para apoyar la OT1-2A • pMig II construcción, el Dr. Juan Carlos Zúñiga Pflucker (Departamento de Inmunología de la Universidad de Toronto) para el apoyo a la línea celular OP9-DL1, y el Dr. Kent Vrana E (Departamento de Farmacología de la Universidad de Penn State College of Medicine) para ayudar al diseño de este estudio. Este proyecto está financiado, en virtud de concesiones con el número de concesión K18CA151798 del Instituto Nacional del Cáncer, la Fundación Barsumian y el Melanoma Research Foundation (canción J.).

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-primed,+in+vitro-activated+CD4++effector+T+cells+establish+long-term+memory+without+exogenous+cytokine+support+or+ongoing+antigen+exposure.">Tumor-primed, in vitro-activated CD4+ effector T cells establish long-term memory without exogenous cytokine support or ongoing antigen exposure.</a> J. Immunol. 184, 5612-5618 (2010).</li><li>Hinrichs, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+effector+CD8++T+cells+derived+from+naive+rather+than+memory+subsets+possess+superior+traits+for+adoptive+immunotherapy.">Human effector CD8+ T cells derived from naive rather than memory subsets possess superior traits for adoptive immunotherapy.</a> Blood. 117, 808-814 (2011).</li><li>Alajez, N. M., Schmielau, J., Alter, M. D., Cascio, M., Finn, O. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+a+tumor-specific,+MHC-unrestricted+T-cell+receptor+expressed+on+effector+cells+of+the+innate+and+the+adaptive+immune+system+through+bone+marrow+transduction+and+immune+reconstitution.">Therapeutic potential of a tumor-specific, MHC-unrestricted T-cell receptor expressed on effector cells of the innate and the adaptive immune system through bone marrow transduction and immune reconstitution.</a> Blood. 105, 4583-4589 (2005).</li><li>Yang, L., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+provision+of+antigen-specific+T+cell+immunity+by+programming+hematopoietic+stem+cells.">Long-term in vivo provision of antigen-specific T cell immunity by programming hematopoietic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 4518-4523 (2005).</li><li>Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrathymic+generation+of+tumor-specific+T+cells+from+genetically+engineered+human+hematopoietic+stem+cells+via+Notch+signaling.">Extrathymic generation of tumor-specific T cells from genetically engineered human hematopoietic stem cells via Notch signaling.</a> Cancer Res. 67, 2425-2429 (2007).</li><li>Boztug, K., Med, N. .. E. n. g. l. .. J. .. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem-cell+gene+therapy+for+the+Wiskott-Aldrich+syndrome.">Stem-cell gene therapy for the Wiskott-Aldrich syndrome.</a> N. Engl. J. Med. 363, 1918-1927 (2010).</li><li>Peerani, R., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enabling+stem+cell+therapies+through+synthetic+stem+cell-niche+engineering.">Enabling stem cell therapies through synthetic stem cell-niche engineering.</a> J. Clin. Invest. 120, 60-70 (2010).</li><li>Mendez-Ferrer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+and+haematopoietic+stem+cells+form+a+unique+bone+marrow+niche.">Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.</a> Nature. 466, 829-834 (2010).</li><li>Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+the+generation+of+patient-specific+pluripotent+stem+cells+for+combined+gene+and+cell+therapy+of+hematologic+disorders.">Towards the generation of patient-specific pluripotent stem cells for combined gene and cell therapy of hematologic disorders.</a> Hematology Am. Soc. Hematol. Educ. Program. , 17-22 (2007).</li><li>Boitano, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aryl+hydrocarbon+receptor+antagonists+promote+the+expansion+of+human+hematopoietic+stem+cells.">Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells.</a> Science. 329, 1345-1348 (2010).</li><li>Himburg, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotrophin+regulates+the+expansion+and+regeneration+of+hematopoietic+stem+cells.">Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells.</a> Nat. Med. 16, 475-482 (2010).</li><li>Tanigaki, K., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+lymphocyte+development+by+Notch+signaling.">Regulation of lymphocyte development by Notch signaling.</a> Nature immunology. 8, 451-456 (2007).</li><li>Zhao, T., Zhang, Z. N., Rong, Z., Xu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenicity+of+induced+pluripotent+stem+cells.">Immunogenicity of induced pluripotent stem cells.</a> Nature. 474, 212-215 (2011).</li></ol>

No hay conflictos de interés declarado.

<html> Para ACT terapias basadas en la generación in vitro de un gran número de Ag altamente reactivos específicos de las células T in vivo para la re-perfusión es una solución óptima. Aunque nuestro método in vitro da lugar funcional de las células T a partir de células iPS, un gran número de células iPS derivadas de las células mueren en cuatro semanas, sobre todo en la cuarta semana. Se concluye que las señales de supervivencia de señalización Notch mediada por el DL1 así co...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> C57BL/6J ratones </td><td> Jackson Laboratory </td><td> 000664 </td></tr><tr><td> B6.129S7-Rag1 tm1Mom / J </td><td> Jackson Laboratory </td><td> 002216 </td></tr><tr><td> Anti-CD3 (2C11) anticuerpo </td><td> BD PharMingen </td><td> 553058 </td></tr><tr><td> Anti-CD28 (37,51) de anticuerpos </td><td> BD PharMingen </td><td> 553295 </td></tr><tr><td> Anti-CD3 (17A2) de anticuerpos </td><td> BioLegend </td><td> 100202 </td></tr><tr><td> Los anti-CD4 (GK1.5) de anticuerpos </td><td> BioLegend </td><td> 100417 </td></tr><tr><td> Anti-CD8 (53-6,7) de anticuerpos </td><td> BioLegend </td><td> 100714 </td></tr><tr><td> Anti-CD25 (3C7) de anticuerpos </td><td> BioLegend </td><td> 101912 </td></tr><tr><td> Unti-CD44 (1M7) de anticuerpos </td><td> BioLegend </td><td> 103012 </td></tr><tr><td> Anti-CD117 (2B8) de anticuerpos </td><td> BioLegend </td><td> 105812 </td></tr><tr><td> Anti-TCR-β (H57597) de anticuerpos </td><td> BioLegend </td><td> 109220 </td></tr><tr><td> Anti-IL-2 (JES6-5H4) anticuerpo </td><td> BioLegend </td><td> 503810 </td></tr><tr><td> Anti-IFN-γ (XMG1.2) de anticuerpos </td><td> BioLegend </td><td> 505822 </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> ABCD1234 </td></tr><tr><td> α-MEM </td><td> Invitrogen </td><td> A10490-01 </td></tr><tr><td> FBS </td><td> HyClone </td><td> SH3007.01 </td></tr><tr><td> Un Brefeldin </td><td> Sigma </td><td> B7651 </td></tr><tr><td> Polybrene </td><td> Sigma </td><td> 107689 </td></tr><tr><td> GeneJammer </td><td> Integrado de Ciencias </td><td> 204130 </td></tr><tr><td> ARN kit &lt;/ Td&gt; <td> Qiagen </td><td> 74104 </td></tr><tr><td> ADN kit </td><td> Qiagen </td><td> 69504 </td></tr><tr><td> CD8 kit de aislamiento </td><td> Miltenyi Biotec </td><td> 130-095-236 </td></tr><tr><td> Tampón de lisis ACK </td><td> Lonza </td><td> 10-548E </td></tr><tr><td> mFlt-3L </td><td> PeproTech </td><td> 250-31L </td></tr><tr><td> mIL-7 </td><td> PeproTech </td><td> 217-17 </td></tr><tr><td> Gelatina </td><td> Sigma </td><td> G9391 </td></tr><tr><td> FITC anti-OVA anticuerpo </td><td> Rockland Immunochemicals </td><td> 200-4233 </td></tr><tr><td> Permeabilización de amortiguación </td><td> BioLegend </td><td> 421002 </td></tr><tr><td> BSA </td><td> Sigma </td><td> A7906 </td></tr><tr><td> Formaldehído </td><td> Sigma </td><td> F8775 </td></tr><tr><td> 0,4 m de filtro </td><td> Millipore </td><td></td></tr><tr><td&gt; MoFlo clasificador de células </td><td> Dake Cytomation </td><td></td></tr><tr><td> Calibur citómetro de flujo </td><td> BD </td><td></td></tr><tr><td> LSR II citómetro de flujo </td><td> BD </td><td></td></tr><tr><td> Ratón inmovilización </td><td> Braintree Científico </td><td></td></tr></tbody></table>

directed differentiation of induced pluripotent stem cells towards t lymphocytes

La transferencia de células adoptivo (ACT) de antígenos específicos de células CD8 + linfocitos T citotóxicos (CTL) es un tratamiento prometedor para una variedad de tumores malignos 1. CTL puede reconocer las células malignas mediante la interacción con los antígenos tumorales receptores de células T (TCR), y citotoxinas liberación de citoquinas, así como para matar las células malignas. Se sabe que menos disociados y central-memoria como-(denominado altamente reactivo) CTL son la población óptima para ACT basado en inmunoterapia, porque estos CTL tienen un potencial proliferativo alta, son menos propensos a la apoptosis que las células más diferenciadas y tienen un mayor capacidad para responder a las citoquinas homeostáticos 2-7. Sin embargo, debido a las dificultades en la obtención de un elevado número de linfocitos T citotóxicos tales de los pacientes, hay una necesidad urgente de encontrar un nuevo enfoque para generar altamente reactivos Ag-CTLs específicos para el éxito a base de ACT-terapias. TCR transducción del vástago auto-renovablecélulas para la reconstitución inmune tiene un potencial terapéutico para el tratamiento de enfermedades 8-10. Sin embargo, el enfoque para obtener células madre embrionarias (CME) de los pacientes no es factible. Aunque el uso de células madre hematopoyéticas (HSC) con fines terapéuticos ha sido ampliamente aplicado en la clínica de 11-13, las CMH se han reducido la capacidad de diferenciación y proliferación, y las CMH son difíciles de crecer en cultivo celular in vitro 14-16. La tecnología reciente de células iPS y el desarrollo de un sistema in vitro para la entrega de genes son capaces de generar células iPS de pacientes sin ningún tipo de abordaje quirúrgico. Además, como CME, las células iPS poseen la capacidad proliferativa indefinida in vitro, y se ha demostrado que se diferencian en células hematopoyéticas. De este modo, las células iPS tienen un mayor potencial para ser utilizado en el ACT basado en inmunoterapia en comparación con los CES o HSCs. Aquí, se presentan métodos para la generación de linfocitos Tcitos de las células iPS in vitro y en vivo de la programación de antígenos específicos de linfocitos T citotóxicos de las células iPS para la promoción de la vigilancia del cáncer inmunológico. La estimulación in vitro con un ligando muesca impulsa la diferenciación de células T a partir de células iPS, y TCR resultados de transducción de genes en células iPS diferenciadores en antígenos específicos de células T in vivo, lo cual impide el crecimiento del tumor. De este modo, demostramos antígeno específico de la diferenciación de células T a partir de células iPS. Nuestros estudios proporcionan un enfoque potencialmente más eficientes para la generación de antígenos específicos de linfocitos T citotóxicos para las terapias basadas en ACT y facilitar el desarrollo de estrategias terapéuticas para las enfermedades.

Watch this Scientific Journal Video about Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes at JoVE.com

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Generación de linfocitos T de madre pluripotentes inducidas (iPS) a las células da un enfoque alternativo de la utilización de células madre embrionarias para células T basada en la inmunoterapia. El método muestra que mediante la utilización de cualquiera<em&gt; In vitro</em&gt; O<em&gt; En vivo</em&gt; Sistema de inducción, las células iPS son capaces de diferenciarse en los linfocitos T, tanto convencionales como antígeno específico.

Diferenciación dirigida de células madre pluripotentes inducidas hacia los linfocitos T

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

directed-differentiation-induced-pluripotent-stem-cells-towards-t

Research

JoVE Journal

Immunology and Infection

18.4K vistas.  Pennsylvania State University College of Medicine. Generación de linfocitos T de madre pluripotentes inducidas (iPS) a las células da un enfoque alternativo de la utilización de células madre embrionarias para células T basada en la inmunoterapia. El método muestra que mediante la utilización de cualquiera In vitro O En vivo Sistema de inducción, las células iPS son capaces de diferenciarse en los linfocitos T, tanto convencionales como antígeno específico.

Video: Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Métodos para evaluar la Beta mediada por la muerte celular por linfocitos T citotóxicos

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Investigación

Inmunología e Infección

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

La expansión de los linfocitos T citotóxicos sangre del cordón umbilical que Citomegalovirus Target, Virus de Epstein-Barr, adenovirus y

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

La generación de células T reguladoras inducidas de células humanas primarias T vírgenes y de memoria

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

La transducción adenoviral de células T CD4 + naïve a estudiar la diferenciación Treg

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Aislamiento y diferenciación de Th17 Naïve linfocitos T CD4

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Derivación de células T<em&gt; In Vitro</em&gt; De células madre embrionarias de ratón

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

Diferenciación in vitro de las células CD4 + humano FOXP3 + células T reguladoras (Induced iTregs) de T naive CD4 + células utilizando un protocolo de TGF-β que contienen

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

La cuantificación rápida de Blastogénesis inducida por mitógeno de linfocitos T para identificar fármacos inmunomoduladores

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Un modelo de ratón In Vivo a medida ingenuo CD4 células T activación, proliferación y diferenciación de Th1 inducida por las células dendríticas derivadas de la médula ósea

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

Diferenciación inducida de células similares a células M en monocapas de enteroide ileal derivadas de células madre humanas

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...