SS é apoiado em parte pela Med HHMI em Grad Formação Grant através do Programa TBMM. MHW é suportado, em parte, por um prêmio de desenvolvimento de carreira do Departamento de Assuntos de Veteranos eo generoso apoio do Dr. Harold e Sra. M. Selzman. Este trabalho também foi apoiado em parte por linfoma NIH SPORE concessão P50CA126752 e R01 NIH DK093660.

Dia 0 1. Isolamento de PBMCs a partir de Sangue Humano <ol><li> Recolher 20 ml de sangue humano fresco, utilizando a punção venosa em tubos Vacutainer Na-heparina. </li><li> Mix de sangue e Avançado RPMI 1640 em 01:01 (v / v) a relação. </li><li> Adicionam-se 20 ml de meio de Lymphoprep num tubo de centrifugação de 50 ml (25 ° C). Lentamente camada 25-30 ml de RPMI-1640 de sangue mistura no topo da Lymphoprep. </li><li> Centrifugar a 400 xg durante 40 min sem freios. </li><li> Recolher as duas camadas distintas e difusa utilizando uma pipeta descartável, em 10 ml de PBS 1x (25 ° C) e perfazer o volume até 50 ml com PBS 1x. </li><li> Centrifugar a 450 xg durante 10 min. </li><li> Aspirar o sobrenadante completamente e adicione 20 ml de RPMI 1640 Avançado. </li><li> Centrifugar a 400 xg durante 5 min. </li><li> Aspirar o sobrenadante. Adicionar 10 mL de meio de células T completo suplementado com 5 ng / ml de rhIL-15 pré-aquecido a 37 ° C. </li><li> Contar o número de células e a placa de cultura de uma camada de tecido de 24 poçosed placa a 2 x 10 6 células / cavidade em meios completos de células T suplementado com 5 ng / ml de rhIL-15 Adiciona-se água estéril para poços adjacentes. Incubar durante a noite numa incubadora humidificada a 37 ° C, 5% CO 2. </li></ol> Dia 1 2. Placas de revestimento com anticorpo anti-CD28 e anti-CD3 para estimular as células T <ol><li> Diluir os anticorpos anti-CD28 e anti-CD3 em água estéril a uma concentração de 1 ug / ml cada um. </li><li> Adicionar 500 ul de solução de anticorpo a cada 5 poços marcados de uma cultura de tecido não revestido 24 placa bem. </li><li> Adicionar água estéril para descansar dos poços. Enrole a placa no envoltório do psiquiatra e coloque na geladeira a 4 ° C. </li></ol> 3. Nucleofection de não estimuladas Células T <ol><li> Pré-aquecer media de células T a 37 ° C e com suplemento de 5 ng / ml de rhIL-15. Preparar a solução de Nucleofector completa por adição de 500 ul de suplemento Nucleofector 1-2,25 ml de solução de Nucleofector. </li><li> Aug liquot 5 cada um dos transposões (Zeo-pT-CMV-eGFP) e transposase (pCMV-piggyBac) num tubo de microcentrífuga de 1,5 ml Nota:. pode ser necessário optimizar a transposase e transposon quantidade de ADN para entrega do gene óptima e para minimizar toxicidade celular. Os plasmídeos podem ser obtidos a partir dos autores, a pedido. </li><li> PBMCs de colheita a partir da placa de cultura de tecido 24 bem para um tubo de 50 ml. Contar o número de células. Salvar 2 x 10 6 células para utilização como controlo durante a citometria de fluxo. </li><li> Adicionar 7-10 x 10 6 células para um tubo de 15 ml e centrifugar a 400 xg, 5 min, o sobrenadante aspirado e dedo-flick o sedimento. </li><li> Adicionar 100 ul de solução de T nucleofection célula completa para afrouxado pellet celular. </li><li> Adicionar a mistura de solução de células para o tubo contendo os plasmídeos. </li><li> Adicionar a mistura de solução de células-plasmídeo para a parte inferior da tina nucleofection. Tenha cuidado para não introduzir quaisquer bolhas. </li><li> Nucleofect as células utilizando program U-014 (células não estimuladas, T humanos, <a href="http://bio.lonza.com/resources/product-instructions/protocols/" target="_blank">http://bio.lonza.com/resources/product-instructions/protocols/</a> ). </li><li> Imediatamente adicionar 500 ul de meio pré-aquecido com rhIL-15 para a cuvete. Transferir as células para um poço de uma placa de 24 poços com 1,5 ml de meio pré-aquecido com rhIL15. </li><li> Incubar durante a noite numa incubadora humidificada a 37 ° C, 5% CO 2. </li></ol> Dia 2 4. Estimulação não específica das células T <ol><li> Colheita de células e determinar o número de células. Separe 0,5 x 10 6 células por citometria de fluxo para determinar a frequência de células positivas para GFP-. Use as CMSPs não transfectadas como controlo. </li><li> Aspirar a solução de anticorpo a partir do CD3/28 placa de cultura de tecido não revestido e lave cada cavidade com meio de células T. </li><li> Ressuspender as células em nucleofected 0,5 x 10 6 células por ml em meio de CTL completassuplementado com 5 ng / ml de rhIL-15. Adicionar 2,0 ml de cada uma das células nucleofected em quatro cavidades da placa de cultura de tecido não. Adicionar 0,5 x 10 6 células não nucleofected para o dia 5 de bem. </li><li> Incubar durante 3 dias numa incubadora humidificada a 37 ° C, 5% CO 2. </li></ol> Dia 5 <ol><li> Colheita das células T estimuladas a partir da placa de cultura de tecido não revestido. </li><li> Contagem e replate as células numa placa de cultura de 24 poços revestidos com tecido menos 0,7 x 10 6 células / ml em meio de células T com 5 ng / ml de IL-15. </li></ol> Dia 7 7. Análise da Expressão do Gene <ol><li> Células de colheita e coram com anti-anticorpo humano CD8 (poderia também usar anti-CD3 e anti-CD4) e análise por citometria de fluxo para a expressão de GFP%. </li></ol> Dia 8 (Opcional) 8. Expansão de células T <ol><li> No dia 8 após a transfecção, as células T podem ser novamente plaqueadas em T-Cell media com IL-15 a uma densidade de 0,7 x 10 6 células por poço de uma nova expansão 16. </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PiggyBac+Transposon-mediated+Gene+Transfer+in+Human+Cells.">PiggyBac Transposon-mediated Gene Transfer in Human Cells.</a> Mol. Ther. 15 (1), 139 (2007).</li><li>Nakazawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+PiggyBac+transposon+system+for+the+sustained+genetic+modification+of+human+T+lymphocytes.">Optimization of the PiggyBac transposon system for the sustained genetic modification of human T lymphocytes.</a> J. Immunother. 32 (8), 826 (2009).</li><li>Raja Manuri, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac+transposon/transposase+system+to+generate+CD19-specific+T+cells+for+treatment+of+B-lineage+malignancies.">piggyBac transposon/transposase system to generate CD19-specific T cells for treatment of B-lineage malignancies.</a> Hum. Gene Ther. 21 (4), 427 (2010).</li><li>Doherty, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperactive+piggyBac+gene+transfer+in+human+cells+and+in+vivo.">Hyperactive piggyBac gene transfer in human cells and in vivo.</a> Hum. 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Ther. 18 (4), 1531 (2010).</li><li>Galvan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mapping+of+PiggyBac+transposon+integrations+in+primary+human+T+cells.">Genome-wide mapping of PiggyBac transposon integrations in primary human T cells.</a> J. Immunother. 32 (8), 837 (2009).</li><li>Nakazawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PiggyBac-Mediated+Cancer+Immunotherapy+Using+EBV-Specific+Cytotoxic+T-Cells+Expressing+HER2-Specific+Chimeric+Antigen+Receptor.">PiggyBac-Mediated Cancer Immunotherapy Using EBV-Specific Cytotoxic T-Cells Expressing HER2-Specific Chimeric Antigen Receptor.</a> Mol. Ther. 19 (12), 2133 (2011).</li><li>Huye, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+mTor+Inhibitors+With+Rapamycin-resistant+T+Cells:+A+Two-pronged+Approach+to+Tumor+Elimination.">Combining mTor Inhibitors With Rapamycin-resistant T Cells: A Two-pronged Approach to Tumor Elimination.</a> Mol. Ther. 19 (12), 2239 (2011).</li><li>Vera, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+production+of+antigen-specific+T+cells+for+preclinical+and+clinical+applications+using+gas-permeable+rapid+expansion+cultureware+(G-Rex).">Accelerated production of antigen-specific T cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (G-Rex).</a> J. Immunother. 33 (3), 305 (2010).</li><li>Kahlig, K. 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Não há conflitos de interesse declarados.

O método aqui descrito permite a modificação de transgene estável primárias linfócitos T humanos. Temos anteriormente testado o uso do sistema de transposon piggyBac para modificar as células T para expressar um gene repórter (por mais de 4 semanas), um gene suicida não imunogénica, um receptor do antigénio quimérico para uma imunoterapia adoptiva (por mais de 100 dias), e de projetar a resistência a medicamentos imunossupressores 7,13-15. Non-viral modificação de células T para a imu...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Lympholyte </td><td> Cedarlane </td><td> CL5015 </td><td></td></tr><tr><td> Avançado RPMI 1640 </td><td> LifeTechnologies </td><td> 12633020 </td><td></td></tr><tr><td> De soro fetal bovino Hyclone </td><td> Fisher Scientific </td><td> SH3008803 </td><td></td></tr><tr><td> GlutaMAX-I Suplemento </td><td> LifeTechnologies </td><td> 35050-061 </td><td></td></tr><tr><td> IL-15 humana recombinante Protein </td><td> eBioscience </td><td> 14-8159 </td><td></td></tr><tr><td> EndoFree Plasmid Maxi Kit </td><td> Qiagen </td><td> 12362 </td><td></td></tr><tr><td> Amaxa Nucleofector </td><td> Lonza </td><td> AAD-1001S </td><td> &amp; Nbsp; </td></tr><tr><td> Human T Cell do Nucleofector Kit </td><td> Lonza </td><td> VPA-1002 </td><td></td></tr><tr><td> CD8-APC </td><td> Southern Biotech </td><td> 9536-11 </td><td></td></tr><tr><td> Anti-CD3 Humano </td><td> eBioscience </td><td> 16-0037-81 </td><td></td></tr><tr><td> Anti-CD28 humano </td><td> BD Pharmingen </td><td> 555725 </td><td></td></tr><tr><td> Placa 24 Cultura de Tecidos Bem tratada </td><td> BD Falcon </td><td> 353047 </td><td></td></tr><tr><td> 24 Cultura de Tecidos Bem Não Tratada Placa </td><td> BD Falcon </td><td> 351147 </td><td></td></tr><tr><td></td><td></td><td></td><td> Completa de células T mídia composição 1x avançada RPMI 1640 5% de calor soro bovino fetal inactivado 2 mM GlutamaxIM-I </td></tr></tbody></table>

piggybac transposon system modification of primary human t cells

O piggyBac sistema transposon é naturalmente ativo, originalmente derivado da 1,2 repolho traça looper. Este sistema não-viral é o plasmídeo com base, mais comumente utilizando dois plasmídeos com uma expressão da enzima transposase piggyBac e um transposon plasmídeo que alberga o gene (s) de interesse entre os elementos de repetição invertidos, que são necessários para a actividade de transferência de genes. PiggyBac medeia a transferência de genes através um &quot;corta e cola&quot; o mecanismo através do qual se integra o segmento transposase do transposão no genoma da célula alvo (s) de interesse. piggyBac demonstrou actividade a entrega eficiente de genes em uma ampla variedade de 1,2 insectos, mamíferos 3-5, e humana cells6 incluindo células T primárias humanos 7,8. Recentemente, uma transposase piggyBac hiperactivo foi gerada a melhoria da eficiência de transferência de genes 9,10. Os linfócitos T humanos são de intere clínicoª para a imunoterapia adoptiva de câncer 11. De nota, o primeiro ensaio clínico envolvendo transposon modificação de células T humanas utilizando o sistema de transposon Sleeping beauty foi aprovado 12. Temos anteriormente avaliaram a utilidade de piggyBac como metodologia não virais para a modificação genética de células T humanas. Encontrámos piggyBac ser eficientes na modificação genética de células T humanas com um gene repórter e um não-imunogénico gene suicida induzível 7. Análise de locais de integração genómicos revelou uma falta de preferência para integração em ou perto de proto-oncogenes conhecidos 13. Usamos a piggyBac gene-modificam linfócitos T citotóxicos para transportar um receptor do antigénio quimérico dirigido contra o antigénio de tumor HER2, e descobriram que as células T modificadas gene-alvo mediada por morte de HER2-positivos de células tumorais in vitro e in vivo num modelo de rato ortotópico 14. Temos também utilizado piggyBacpara gerar células T humanas resistentes a rapamicina, que devem ser úteis em terapias de cancro em que a rapamicina é utilizada 15. Relata-se um método para utilizar piggyBac para modificar geneticamente células T primárias humanas. Isto inclui o isolamento de células mononucleares de sangue periférico (PBMCs) a partir de sangue humano, seguido por cultura, a modificação do gene, e a activação das células T. Para efeitos do presente relatório, as células T foram modificados com um gene repórter (eGFP), para análise e quantificação da expressão de genes através de citometria de fluxo. PiggyBac pode ser usado para modificar células T humanas com uma variedade de genes de interesse. Apesar de termos usados ​​piggyBac para dirigir as células T a antigénios de tumor 14, também foram utilizados piggyBac para adicionar um interruptor de segurança induzível de modo a eliminar as células de genes modificados, se necessário 7. A capacidade de carga de grande piggyBac também permitiu a transferência de genes deuma grande molécula de rapamicina mTOR resistente (15 kb) 15. Por conseguinte, apresenta-se uma metodologia não-viral para estável gene modificação de células T primárias humanas para uma grande variedade de finalidades.

Um diagrama esquemático mostrando as etapas na modificação genética de linfócitos T humanos com um gene repórter (eGFP) é mostrado na Figura 1. Estes plasmídeos estão disponíveis mediante solicitação dos autores. A schemtic demonstrando as etapas geneticamente modificados linfócitos T humanos com um gene repórter (eGFP) é showin na Figura 2. É necessário para activar as células T, a fim de levá-los a dividir, expandir e propagar em cultura. Modificados células T huma...

Watch this Scientific Journal Video about piggyBac Transposon System Modification of Primary Human T Cells at JoVE.com

piggyBac Transposon System Modification of Primary Human T Cells

Descreve-se um método para modificar geneticamente células T primárias humanas com um transgene, utilizando o não-viral<em&gt; PiggyBac</em&gt; Sistema de transposon. As células T modificado para utilizar o<em&gt; PiggyBac</em&gt; Transposon sistema mostra a expressão do transgene estável.

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 ...

piggybac-transposon-system-modification-of-primary-human-t-cells

Research

JoVE Journal

Immunology and Infection

piggyBac Transposon System Modification of Primary Human T Cells

17.1K visualizações.  Baylor College of Medicine. Descreve-se um método para modificar geneticamente células T primárias humanas com um transgene, utilizando o não-viral PiggyBac Sistema de transposon. As células T modificado para utilizar o PiggyBac Transposon sistema mostra a expressão do transgene estável.

Vídeo: piggyBac Transposon System Modification of Primary Human T Cells

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Expansão do sangue periférico humano γδ células T usando zoledronato

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Imunologia e Infecção

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

Determinando a atividade citotóxica Optimal específicos de células CD8 Her2neu Humanos T, comparando a Cr51 Ensaio de Libertação do Sistema xCELLigence

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

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.

Geração de Células T reguladoras induzidas a partir de células T humanas primárias Naïve e Memória

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

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Avaliando a inata Sensing de HIV-1 CD4 Infected<sup&gt; +</sup&gt; As células T por células dendríticas plasmocitóides Usando um<em&gt; Ex vivo</em&gt; Co-cultura do sistema.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/&#955;pir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

Identificação de novos genes associados com alginato de Produção em<em&gt; Pseudomonas aeruginosa</em&gt; Usando Mini-<em&gt; Himar1</em&gt; Mutagênese Mariner Transposon mediada

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Geração de Células T humanas alo-específicos a partir de Sangue Periférico

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

Avaliação da Interface sináptica de células T humano primário do sangue periférico e tecido linfoide

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Infecção pneumocócica de células endotélias humanas primárias em fluxo constante

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

Produção de células CAR-T projetadas por CRISPR humanas

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with ...

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.