Este trabalho foi suportado por concessões do NIH (5U01CA137070) ea Sociedade Americana do Câncer (RSG-09-070-01-LIB), a Cancer Research Institute Investigator Award (MK), um American Heart Association comunhão pré-doutoramento (KM) e um Ministério espanhol da Educação e Ciência Fellowship (APG). Gostaríamos que a agradecer ao Dr. Nicholas Restifo e Dr. Zhiya Yu (NIH / NCI) para sugestões úteis para o protocolo.

1. Prepare Construct Retroviral <ol><li> Sub-clone do T-cell receptor gene (TCR) de interesse em um vetor retroviral (Figura 1, os vetores exemplo PMSG 7, pMIGII 5,8, pMXs de Cellbiolabs). A TCR α e gene da cadeia β deve estar no mesmo vetor sob o controle do mesmo promotor para garantir a expressão igual. Se o TCR de interesse é humano, os domínios humanos constante necessidade de ser substituído por domínios do mouse constante 9. Nossa observação é que TCRs comprimento total humanos não expressar de forma estável no mouse células-T. </li><li> Prepare DNA de alta qualidade utilizando plasmídeo Maxi Kit Qiagen Prep ou produto similar. A concentração do plasmídeo deve ser em torno de 1 mg / mL. </li></ol> 2. Transfecção de células T e de isolamento Dia -1 (células embalagem Plate) <ol><li> Desalojar Platinum E-(Plat-E, Cellbiolabs) células embalagem retroviral com tripsina-EDTA e neutralizar com DMEM mídia. </li><li> Centrifugar as células a 1000 xg por 5 min. Aspirar o sobrenadante e ressuspender o pellet celular em DMEM mídia. </li><li> Determinar o número de células e diluir as células de 0.6x10 6 mL /. ML placa, 10 de suspensão de células em uma placa de cultura 10 milímetros poli-lisina revestido de tecido e crescer durante a noite em uma cela incubadora a 37 ° C, 5% CO 2. </li></ol> Dia 0 (transfecção) <ol start="4"><li> Na manhã seguinte, examine as células em um microscópio de luz. As células devem ser aproximadamente 80% confluentes. </li><li> Remova cuidadosamente os meios de comunicação da placa. Lave as células uma vez com PBS 1x e adicionar 10 mL pré-aquecido, fresca DMEM mídia sem penicilina-estreptomicina (Pen / Strep). (Nota: Pen / Strep podem interferir com a transfecção) </li><li> Prepare complexo transfecção. Preparar a mistura A e B da seguinte forma: <table border="0"><tbody><tr><td> Uma mistura: DNA plasmidial </td><td> 9 mg </td></tr><tr><td> PCL-Eco helper plasmídeo </td><td> 6,3 mg </td></tr><tr><td> OptiMEM </td><td> 1,5 mL </td></tr><tr><td colspan="2"></td></tr><tr><td> Mix B: Lipopfectime 2000 </td><td> 60 mL </td></tr><tr><td> OptiMEM </td><td> 1,5 mL </td></tr></tbody></table> Incubar A e B separadamente por 5 minutos em temperatura ambiente Mix A e B juntos suavemente e incubar por 20 minutos em temperatura ambiente para formar complexos transfecção. </li><li> Gotejamento lentamente a mistura (de 3 mL total) para as células Plat-E. Agite levemente o prato para trás e para distribuir a mistura de transfecção uniformemente. </li><li> Incubar as células a 37 ° CO C / 5 2% em uma incubadora de celular. </li><li> Depois de 6-8 horas, substituir média com 10 mL de meio DMEM fresco (com Pen / Strep) para a produção viral. </li></ol> Placa de revestimento com anticorpos <ol start="10"><li> Rato células-T precisam ser ativados antes de transdução viral. Há uma variedade maneiras para ativar as células-T. Aqui usamos placa ligado anticorpos anti-CD28 CD3e e anti-. Prepare uma mistura de anticorpos de 1 mg / mL de anti-CD3e e 2 mg / mL de anti-CD28 em PBS estéril. </li><li> Dispense 250 L da mistura de anticorpos em cada poço de uma placa de cultura de 24 poços de tecido. A placa pode ser revestido durante a noite a 4 horas ou 2 ° C a 37 ° C. </li></ol> Preparação de uma única célula de suspensão de baço de rato <ol start="12"><li> Baço colheita mouse e transferência para meio RPMI. </li><li> Coloque o baço em um coador de celular. Mash o baço com um êmbolo da seringa em uma placa de cultura 5 centímetros de tecido. Enxágüe células fora do filtro de células com PBS esterilizado. </li><li> Centrífuga 1000 xg, 5 min. </li><li> Elimine o sobrenadante. Ressuspender pellet celular em tampão de lise ACK (2 mL por baço). Incubar 2-3 min à temperatura ambiente. Adicionar meio RPMI até 20 mL e centrifugar 1000 xg por 5 min. </li><li> Elimine o sobrenadante. Ressuspender celular em PBS estéril. Determinar o número de células e xg centrífuga 1000 para 5 min a 4 ° C. Proceder ao isolamento de células-T. </li></ol> Isolamento e ativação de células T do mouse <ol start="17"><li> Magneticamente rótulo as células de acordo com o manual do produto (CD8 + kit de isolamento de células T de Miltenyi Biotec). </li><li> Colocar uma coluna LS (Miltenyi Biotec), no magnetic campo. Aplicar suspensão de células marcado para a coluna. Colete de fluxo (enriquecido rato CD8 + T-cells). Lavar a coluna três vezes com 3 mL de tampão de acordo com o manual do produto e combinar com o anterior por escoamento. </li><li> Mancha as células com anticorpos anti-CD3e e anti-CD8a e analisar por citometria de fluxo (Figura 2a). Para uma separação típica, ~ 80-10% do total de células podem ser isoladas. ~ 90% das células CD8 + são isoladas células-T. </li><li> Centrifugar as células a 1000 xg, 5 min. Desprezar o sobrenadante. Ressuspender o pellet celular em meio RPMI com recombinante humana IL-2 (rhIL2, 20 ng / mL) em 1x10 6 / mL. </li><li> Pouco antes de adicionar as células, remoção da solução de anticorpos da placa (previamente preparado em 2.11). Enxágüe bem cada com PBS estéril e PBS remover. </li><li> Adicionar 1 mL da suspensão de células em cada poço. Colocar a placa a 37 ° C / 5% CO 2 em uma célula incubadora. </li></ol> 3. (Dia 2 e dia 3) Infecção de células T ativadas <ol><li> Após dois dias de produção de vírus, o meio na placa de células Plat-E deve tornar-se amarelo. Transfira o sobrenadante viral a um tubo de 15 mL cônico. Adicionar 10 mL de meio DMEM fresco para a placa para a produção viral mais (opcional). </li><li> Centrífuga sobrenadante vírus em 1000 xg por 5 min para remover detritos células. Transferir cuidadosamente o sobrenadante vírus para um novo tubo. Deixe um pouco de líquido na parte inferior do tubo e não perturbe os restos de células. </li><li> Coletar células T ativadas a partir da placa em um tubo cônico de 50 mL. Determinar o número de células. Salvar algumas células em um tubo separado para ser usado como controle (não-transduzidas) negativo. Centrifugar a 1000 xg por 5 min e descartar o sobrenadante. </li><li> Ressuspender o pellet celular em vírus sobrenadante a 10 6 células por 1 mL com 20 ng / mL rhIL2 e 10 mcg / ml de sulfato de protamina. Adicionar 1 mL de suspensão de células em cada poço de uma placa de 24 poços. Ressuspender as células no tubo de controle em meio RPMI na densidade das células mesmo com a mesma quantidade de sulfato de protamina e rhIL2. Adicionar as células a mesma placa. </li><li> Embrulhe a placa com filme plástico. Centrifugar 90 minutos a 2000 xg, a 32 ° C, sem freio. </li><li> Após a centrifugação, remover o filme plástico. Adicionar 1 mL de meio RPMI fresco com 20 ng / mL rhIL2 a cada poço. Coloque a placa de volta para a incubadora. </li><li> (Opcional) Nível Superior expressão de TCR podem ser alcançados através da coleta do sobrenadante vírus e repetir o procedimento de infecção no dia 3. </li></ol> 4. Expansão das células e Avaliação da Eficiência Transdução <ol><li> Examine o transduzidas células T diariamente. Dividir as células em proporções 01:03, quando necessário, em meio RPMI rhIL2. Não deixe que a overgrow células (média fica amarelo). </li><li> No dia 6 ou 7 dias, mancha as células com o anticorpo e tetrâmero MHC específico para o TCR para avaliar a expressão da TCR em células T de superfície. Use o un-transduzidas células-T como controle (Figura 2c). </li><li> O transduzidas células-T estão prontos para novas aplicações a jusante. </li></ol> 5. Resultados representante Muitas vezes é vantajoso para purificar de células T subconjuntos antes de transdução embora esplenócitos pode ser transduzidas sem purificação. Alta pureza de células T subconjuntos pode ser obtida usando comercial esferas magnéticas (Figura 2a). Para avaliar o nível de expressão de TCRs sobre as células T, anticorpos específicos para TCR alfa ou beta cadeias podem ser usados. Tipicamente 30% a 80% das células podem expressar transduzidas TCR (Figura 2b), dependendo do genes TCR e os títulos de vírus. MHC específico tetrâmero para a TCR também pode ser usado para comprovar expressão funcional de TCRs (Figura 2c). <img src="/files/ftp_upload/2307/2307fig1.jpg" alt="Figura 1" /> Figura 1. Exemplo de células T do gene do receptor construir sub-clonado em um vetor retroviral. Alpha de comprimento total e genes de cadeia beta estão ligadas por uma auto-linker peptídeo clivável 2A 8. <img src="/files/ftp_upload/2307/2307fig2.jpg" alt="Figura 2" /> Figura 2. Exemplo de isolamento bem sucedido e transdução de células T do mouse. (A) células-T CD8 foram isoladas de esplenócitos usando CD8a + T Celular Isolation Kit (Miltenyi Biotec) e corados com anticorpos anti-CD3e e anti-CD8a. Isolado células-T CD8 foram transduzidas com R6C12 TCR específico para humanos gp100: 209 -217 peptídeo 4 e corados com anti-humano Vbeta 8 de anticorpos (b) e HLA-A2kb gp100 tetrâmero (c).

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	<li>Krogsgaard, M., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+T+cells+'see'+antigen.">How T cells 'see' antigen.</a> Nat. Immunol. 6, 239-245 (2005).</li><li>Berg, 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=Antigen/MHC-specific+T+cells+are+preferentially+exported+from+the+thymus+in+the+presence+of+their+MHC+ligand.">Antigen/MHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand.</a> Cell. 58, 1035-1046 (1989).</li><li>Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+antagonist+peptides+induce+positive+selection.">T cell receptor antagonist peptides induce positive selection.</a> Cell. 76, 17-27 (1994).</li><li>Yu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poor+immunogenicity+of+a+self/tumor+antigen+derives+from+peptide-MHC-I+instability+and+is+independent+of+tolerance.">Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance.</a> J. Clin. Invest. 114, 551-559 (2004).</li><li>Holst, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+T-cell+receptor+retrogenic+mice.">Generation of T-cell receptor retrogenic mice.</a> Nat Protoc. 1, 406-417 (2006).</li><li>Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+regression+in+patients+after+transfer+of+genetically+engineered+lymphocytes.">Cancer regression in patients after transfer of genetically engineered lymphocytes.</a> Science. 314, 126-129 (2006).</li><li>Hawley, R. G., Lieu, F. H., Fong, A. Z., Hawley, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+retroviral+vectors+for+potential+use+in+gene+therapy.">Versatile retroviral vectors for potential use in gene therapy.</a> Gene Ther. 1, 136-138 (1994).</li><li>Szymczak, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+multi-gene+deficiency+in+vivo+using+a+single+'self-cleaving'+2A+peptide-based+retroviral+vector.">Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.</a> Nat. Biotechnol. 22, 589-594 (2004).</li><li>Cohen, C. J., Zhao, Y., Zheng, Z., Rosenberg, S. A., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+antitumor+activity+of+murine-human+hybrid+T-cell+receptor+(TCR)+in+human+lymphocytes+is+associated+with+improved+pairing+and+TCR/CD3+stability.">Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability.</a> Cancer Res. 66, 8878-8886 (2006).</li><li>Letourneur, F., Malissen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+a+T+cell+hybridoma+variant+deprived+of+functional+T+cell+receptor+alpha+and+beta+chain+transcripts+reveals+a+nonfunctional+alpha-mRNA+of+BW5147+origin.">Derivation of a T cell hybridoma variant deprived of functional T cell receptor alpha and beta chain transcripts reveals a nonfunctional alpha-mRNA of BW5147 origin.</a> Eur. J. Immunol. 19, 2269-2274 (1989).</li><li>Bendle, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lethal+graft-versus-host+disease+in+mouse+models+of+T+cell+receptor+gene+therapy.">Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy.</a> Nat. Med. 16, 565-570 (2010).</li></ol>

Não há conflitos de interesse declarados.

Várias etapas são fundamentais para alcançar ótimos resultados. Plat-E linhagem de células de embalagens deve ser mantido em condição saudável. Se necessário, as células podem ser várias passagens algumas vezes em meio DMEM com 1 mg / mL puromicina, 10 mcg / ml blasticidin para evitar a perda de expressão retroviral estrutura da proteína. No dia da transfecção, as células devem ser confluentes ~ 80%. Células muito poucos ou muitos podem reduzir o título de vírus. A qualidade do vírus pode ser verificado através de estudos com linhas celulares que podem ser facilmente transduzidas. Desde TCRs exigem CD3 complexos a ser expressa na superfície das células, que usam rotineiramente 58α-/β- células de hibridoma 10 (uma linhagem celular que não expressa endógena TCR α ou β da cadeia, mas se expressa complexo CD3). Obtemos quase 100% de eficiência de transdução de células de hibridoma quando transdução de células com 1 mL do sobrenadante vírus. Finalmente, as células-T precisam ser ativados e proliferando porque retrovírus só pode infectar células em divisão ativa. O método aqui apresentado para a produção de antígenos específicos de células T tem várias limitações. Primeiro, é difícil de alcançar eficiência de transdução de 100% para primário do mouse células-T. Uma parte das células não expressam o TCR de interesse. Segundo, o transduzidas TCR α e cadeias β pode mispair com TCRs endógena 9, que pode reduzir o nível de expressão da TCR de interesse. Além disso, o TCRs mispaired pode causar auto-imunidade in vivo 11. Finalmente, há apenas um prazo limitado a transduzidas células-T pode ser usado. Após cerca de uma semana, as células em apoptose não ser que seja re-estimulado. No entanto, as células podem tornar-se esgotado e perder funções com repetitivas re-estímulos.

Media: DMEM (DMEM calor +10% inativado FBS + Pen / Strep + L-Glu + Piruvato de sódio + ácido não-aminoácidos essenciais). DMEM sem Pen / Strep (Same como meio DMEM, mas sem acrescentar Pen / Strep). Meio RPMI (RPMI calor +10% inativado FBS + L-Glu + ácido não-aminoácidos essenciais + β-Mercaphoethanol + Pen / Strep Piruvato de Sódio +).

retroviral transduction of t-cell receptors in mouse t-cells

Receptores de células T (TCRs) desempenham um papel central no sistema imunológico. TCRs na T-cell superfícies podem especificamente reconhecer antígenos peptídeos apresentados por células apresentadoras de antígenos (APCs) 1. Este reconhecimento leva à ativação de células T e uma série de resultados funcionais (por exemplo, produção de citocinas, matando as células-alvo). Compreender o papel funcional de TCRs é fundamental para aproveitar o poder do sistema imunológico para tratar uma variedade de doenças relacionadas imunologia (por exemplo, câncer ou de auto-imunidade). É conveniente estudar TCRs em modelos de ratos, que pode ser feito de várias maneiras. Fazendo TCR modelos transgênicos do mouse é caro e demorado e, atualmente, existem apenas um número limitado deles disponíveis 2-4. Alternativamente, os camundongos com antígenos específicos de células T podem ser gerados pela medula óssea quimera. Este método também leva várias semanas e requer expertise 5. Transdução retroviral de TCRs em in vitro do mouse ativado células-T é um método rápido e relativamente fácil de obter células-T de especificidade desejada peptídeo-MHC. Antígeno-específica de células T podem ser gerados em uma semana e usado em todas as aplicações a jusante. Estudando transduzidas células-T também tem aplicação direta à imunoterapia humana, como a transferência adotiva de células T humanas transduzidas com antígeno específico TCRs é uma estratégia emergente para o tratamento do câncer 6. Aqui apresentamos um protocolo para a transdução retrovirally TCRs em in vitro do mouse ativado células-T. Ambos os genes humanos e do rato TCR podem ser usados. Retrovírus portadores de genes específicos TCR são gerados e usados ​​para infectar rato células T ativadas com anticorpos anti-CD3 e CD28-anti. Depois de na expansão in vitro, células-T transduzidas são analisados ​​por citometria de fluxo.

Watch this Scientific Journal Video about Retroviral Transduction of T-cell Receptors in Mouse T-cells at JoVE.com

Retroviral Transduction of T-cell Receptors in Mouse T-cells

Nós apresentamos um protocolo para a produção de antígenos específicos do mouse células T usando transdução retroviral

Transdução retroviral de receptor de células T no rato células T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

retroviral-transduction-of-t-cell-receptors-in-mouse-t-cells

Research

JoVE Journal

Immunology and Infection

34.7K visualizações.  New York University School of Medicine. Nós apresentamos um protocolo para a produção de antígenos específicos do mouse células T usando transdução retroviral

Vídeo: Retroviral Transduction of T-cell Receptors in Mouse T-cells

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac modificação do sistema Transposon de células T primárias Humanos

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Imunologia e Infecção

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.

Transdução de adenovírus Naive CD4 células T para estudar a diferenciação 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 ...

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.

Derivação de células T<em&gt; In Vitro</em&gt; De Mouse Células-Tronco Embrionárias

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

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Rato Naïve CD4<sup&gt; +</sup&gt; Isolamento de células T e<em&gt; In vitro</em&gt; Diferenciação em subpopulações de células T

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Transdução retroviral de Células de Medula Óssea progenitoras para gerar células T Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

Gerando<em&gt; De Novo</em&gt; Antígeno-específica T Humano receptores celulares por Transdução retroviral de Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Transdução retroviral de células T helper como uma abordagem genética para estudar mecanismos que controlam a sua diferenciação e função

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

Isolamento das células CD8+ T residentes em rim do rato para análise de citometria de fluxo

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Purificação e Expansão de Células T Assassinos Naturais Invariantes para Estudos In vitro e In vivo

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to assess calcium signaling by band-pass flow cytometry. This protocol is designed to measure calcium responses in primary murine T-cells using full spectrum flow cytometry. Total splenocytes are labeled with the ratiometric calcium indicator dye Indo-1, along with a panel of fluorochrome-conjugated antibodies to cell surface molecules. Leveraging the capabilities of full spectrum flow cytometry provides a platform for utilizing a wide array of cell surface stains in combination with Indo-1. Cells are then analyzed in real-time at 37 &#176;C before and after the addition of an anti-CD3 antibody to stimulate the T-cell receptor. After unmixing the spectral signals, the ratio of calcium-bound to calcium-free Indo-1 is calculated and can be visualized over time for each gated population of splenocytes. This technique can allow for the simultaneous analysis of calcium responses in multiple cell populations.

Calcium influx, a measure of T-cell signaling, is an effective way to analyze responses to T-cell receptor stimulation. This protocol for multiplexing Indo-1 with panels of antibodies directed at cell surface molecules takes advantage of the highly flexible capabilities of full spectrum flow cytometry.

Análise do Influxo de Cálcio Induzido por Receptores de Células T em Células T Primárias Murinas por Citometria de Fluxo de Espectro Completo

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to ...