Este trabalho é apoiado por uma assistência de produção de terapias celulares (contrato NIH-NHLBI (HB-10-03) HHSN26820100000C) (CMR), uma Centros Especializados de Terapia Celular Grant NIH-NHLBI uma U54 HL081007 (CMR), uma ASBMT Young Investigator Award (UG e JV), um Fellow da Sociedade Leucemia e Linfoma especial em Prêmio Pesquisa Clínica (UG), e uma Amy Strelzer Scholar Award Manasevit (AML).

1. Nucleofection DC <ol><li> Colheita derivados de monócitos DCs, que foram enriquecidas com a adesão de plástico, cultivadas por cinco dias usando celular Genix Mídia suplementado com IL4 (1000U/ml), GMCSF (800IU/ml) e ainda venceu por 24 horas usando a maturação citocinas IL4 DC (1000U / ml), GMCSF (800IU/ml), IL6100ng/ml, TNF-α 10ng/ml, IL1-β 10ng/ml e PGE2 (1μg/ml) 1, pela ressuspensão gentil com uma pipeta de 3 ml. </li><li> Contar DCs viável usando azul de tripano, transferência para tubos 15ml 3x com nada menos que 0.5x10 6 e não mais do que 2x10 6 células / tubo. </li><li> Centrífuga DCs para 10mins @ 200g. Durante este período pré-aquecer celular Genix meio suplementado com as citocinas maturação DC (DC meios de maturação) - 2ml/well em três poços de uma cultura de tecidos de 12 bem tratado em uma placa de 37 ° C / 5% CO 2 incubadora. </li><li> Uma vez que células terminar spinning, aspirar o sobrenadante e adicionar os plasmídeos DNA relevantes para cada um dos tubos em uma concentração final de DNA 5μg / tubo. Neste caso, adicione o plasmídeo IE1-pp65 ao tubo n º 1, hexon-Penton ao tubo # 2, e EBNA1-LMP2-BZLF1 ao tubo n º 3. </li><li> Ressuspender DCs e DNA com 100μl de solução Amaxa nucloefection, misture bem e transferir para o cubetas nucleofection. </li><li> Cubetas lugar no nucleofector 4D, escolher programa CB150 (Amaxa / Lonza), e pressione o começo. </li><li> Imediatamente após nucleofection adicionar 500μl do 2ml pré-aquecido celular Genix meio de maturação DC para a cubeta, misturar suavemente por pipetagem cima e para baixo 2-3 vezes, e DCs nucleofected transferência para a chapa 12 bem preparados contendo os 1,5 ml restantes de pré-aquecida maturação media DC. Transferência para a 37 ° C / 5% CO 2 incubadora por mais 12-18hrs. </li></ol> 2. Estimulação das células T <ol><li> Colheita e contagem de DCs nucleofected, e irradiar a 30Gy. Lavar uma vez com 10ml de meio de CTL (45% RPMI, Cliques 45% EHAA, 10% FBS, 2mM Glutamax) e @ 3 ressuspender x 10 5 DCs por ml de mídia CTL. </li><li> Piscina um mínimo de 7.5x10 5 (2,5 ml) e um máximo de 15x10 5 (5ml) de DCs contendo cada um dos plasmídeos e transferir os DCs agrupados ao dispositivo G-Rex, que será então colocado na incubadora. </li><li> Para a preparação de células de resposta usar PBMCs anteriormente congelados ou não aderente células mononucleares que permanecem após a seleção DC (adesão ou CD14 seleção). Descongelar as células, a transferência para meio de cultura pré-aquecida, lave uma vez com a mídia CTL. Ressuspender as células em Media CTL, a contagem das células e levá-los a uma concentração de 2x10 6 células por ml. Leve 15x10 6 células ou 7.5ml e suplementar com 30000U IL4 (1000U/ml -. Conc final) e 300ng IL7 (10ng/ml - conc final.). </li><li> Transferência 7.5ml de PBMC (15x10 6 células) para o G-Rex e superior até o biorreator com a mídia CTL para um volume total de 30ml. </li><li> Cultura do G-Rex por 6-7 dias a 37 ° C / 5% CO 2 incubadora umidificada. </li></ol> 3. T expansão celular <ol><li> No dia 07/06, aspirar 10ml de mídia, então misturar as células do 20ml restantes de mídia com uma pipeta de 10 ml e contagem de células viáveis ​​com azul de tripano. Se houver &lt;50x10 6 repor com novas mídias + citocinas. Se houver&gt; 50x10 6 células remover 10ml de suspensão celular, a transferência para um novo G-Rex, e depois alimentar tanto G-Rexs com novas mídias CTL + citocinas. </li><li> Cultura para um adicional de 4-6 dias. Uma vez que células suficientes foram ampliados, realizar uma caracterização fenotípica e funcional do excesso de CTL e criopreservar para uso futuro. </li></ol> 4. Resultados representativos: Um esquema de nosso FDA-approved processo de geração de multivirus específicas CTL é mostrado na Figura 1. Em contraste com protocolos convenção multivirus CTL que usam adenovectors e EBV-LCL para estimular as células-vírus reativa T 2 temos substituído material de vírus infeccioso com plasmídeos DNA que codificam antígenos derivados de cada um dos três vírus. Para estimular a CTL trivirus nós projetamos três multicistronic plasmídeos codificando hexon e Penton de adenovírus, IE1 e pp65 do CMV, e EBNA1, LMP2, e BZLF1 de EBV. Estes antígenos foram escolhidos com base em resultados encorajadores clínica de nossas próprias e de outros grupos mostrando que as células T dirigidos contra Adv-hexon e Penton 2,4-6, e CMV-1E1 e CMV pp65-são protetores in vivo 7. Para EBV, EBNA1 é um CD4 + T imunodominantes antígeno alvo celular expressa em todas as neoplasias malignas associadas ao EBV e em células normais infectadas pelo EBV B 8,9, LMP2 é imunogênica em vários tipos de HLA múltiplas e expressa na maioria dos tumores malignos EBV, 10,11 enquanto BZLF1 codifica uma imunodominantes, antígeno precoce imediato ciclo lítico que estimula tanto CD4 + e CD8 + T da maioria dos indivíduos e é provável que importantes para o control de células replicação do vírus 12. Para otimizar ainda mais os nossos métodos de fabricação que colaborou com Tecnologia Nature que gerou minimizado, sem antibiótico (FDA-compliant) plasmídeos para a estimulação CTL 13,14. Usando esta estratégia que consistentemente alcançar eficiências nucleofection de&gt; 35%, mantendo a viabilidade das células de alta (dados não mostrados) 3. A Figura 2 mostra que a freqüência de células T-vírus específico em resposta a cosmídeos otimizado medida pelo IFNγ ELISPOT, foi maior que em resposta a convencional plasmídeos pShuttle baseado expressão expressando os mesmos antígenos (n = 8 Adenovirus, n = 4 CMV e EBV n = 2). O melhor rácio de DC: PBMC foi importante para a estimulação das células T potente como mostrado na Figura 3, onde uma proporção de 1:50 produzidos sub-ótimo de ativação em comparação com um S 1:20: R ratio (n = 2 doadores). Produção de suficientes números CTL com a especificidade do antígeno larga é um pré-requisito para a eficácia clínica contra os três vírus. Isto é conseguido pela cultura CTL no G-Rex, que apóia a expansão de células T superiores quando comparados com os placas de 24 poços (Figura 4A) 15, enquanto a adição de IL4 e IL7 a aumentos culturas do repertório e especificidade, como mostrado na Figura 4B onde a freqüência de células T reativas contra o CMV-pp65 derivados HLA-A2 RESTRIÇÕES NLV peptídeo foi avaliada em culturas gerado na presença ou ausência de IL4 e / ou IL7 16,17. Para avaliar a capacidade fenótipo e funcional das células expandidas realizamos análise de citometria de fluxo, intracelular de citocinas coloração / IFNγ ELISPOT, e 51 testes de liberação de Cr sobre o produto final para a criopreservação / perfusão. Normalmente as células geradas são policlonais com uma população mista de células CD4 + e CD8 + T com antígeno especificidade detectáveis ​​nos dois compartimentos da célula T. O CTL são capazes de matar antígeno viral expressando as células-alvo, mas não do vírus negativos parcialmente HLA alvos combinados, indicando que eles não devem induzir enxerto-versus-hospedeiro (GVHD) in vivo (Figura 5). <img src="/files/ftp_upload/2736/2736fig1.jpg" alt="Figura 1"/> Figura 1. Rctl protocolo geração. Primeiro, DCs são nucleofected com o antígeno viral codificação de plasmídeos e então misturado com PBMCs autólogo em um R: S de 10 ou 20:1. Células são expandidas no G-Rex por 10-14 dias, na presença de IL4 e IL7, então colhidos, contados, testado para a identidade de função, e esterilidade, e depois criopreservados para uso clínico. <img src="/files/ftp_upload/2736/2736fig2.jpg" alt="Figura 2"/> Figura 2. Cosmídeos Optimized induzem ativação de células T in vitro superiores. DCs foram nucleofected com otimizado, FDA-compliant plasmídeos codificando hexon e Penton (Adv), IE1 e pp65 (CMV), e EBNA1, LMP2, e BZLF1 (EBV) ou plasmídeos pShuttle convencionais de codificação os mesmos antígenos. Estes foram usados ​​para estimular as células T e especificidade foi analisada pelo IFNγ ELISPOT 10 dias pós-estimulação. <img src="/files/ftp_upload/2736/2736fig3.jpg" alt="Figura 3"/> Figura 3 DC ideal:. Proporções de células T para a ativação do CTL. DCs de 2 doadores foram nucleofected com todos os três plasmídeos otimizado e, em seguida, utilizado para estimular PBMCs autólogo em 1:20 ou 1:50 DC: relação de PBMC. A freqüência de células T reativada foi avaliada no dia 10 de IFNγ ELISPOT. <img src="/files/ftp_upload/2736/2736fig4.jpg" alt="Figura 4"/> Figura 4. Expansão de células T no G-Rex usando citocinas melhorando. Um painel mostra o dispositivo G-Rex, bem como aparência CTL na membrana gás permeável, avaliados por microscopia. Uma comparação entre a saída de células alcançado na convenção de cultura de tecidos tratados placas vs G-Rex também é mostrada. Painel B mostra a freqüência de CMV CTL pentâmero positivos alcançados nas culturas expandiu na presença de nenhum de citocinas, IL4 sozinho, IL7 sozinho e IL4 + IL7. <img src="/files/ftp_upload/2736/2736fig5.jpg" alt="Figura 5"/> Figura 5. Fenótipo e função de CTL expandida. Um painel mostra um exemplo representativo do fenótipo do CTL multivirus expandido, que são policlonais com uma mistura de CD4 + (45% - helper) e CD8 + (42% - citotóxicos), as células T, dos quais a maioria (95%) expressou o memória marcador CD45RO + / CD62L + Panel. B mostra que essas células são específicos para todos os antígenos estimulante e são polifuncionais, avaliada pela coloração de citocinas intracelulares para detectar a produção de IFNΓ e TNFa após a estimulação antigênica. Painel C mostra que a CTL expandida são funcionais medida por Cr ensaio 51. Autólogo LCL, sozinho ou transduzidas com um vetor nulo ou um vetor adenoviral expressando-CMV pp65 foram usados ​​como alcatrãofica. Alloreactivity foi avaliada através explosões alogênico PHA como um alvo.

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Juan F. Vera é um consultor de Wilson Manufacturing Wolf. Os outros autores não têm divulgações relevantes.

Infecções virais são responsáveis ​​por significativa morbidade e mortalidade em pacientes que são imunocomprometidos por sua doença ou seu tratamento. Após o transplante, por exemplo, infecções causadas por herpesvírus persistentes, como EBV e CMV, bem como por vírus respiratórios, tais como Vírus Sincicial Respiratório (VSR), são bem conhecidos, enquanto a importância de infecções causadas por Adv, vírus BK, e humanos herpesvirus (HHV) -6 tem mais recentemente sido apreciado. Enquanto agentes farmacológicos são a terapia padrão para algumas infecções, eles têm toxicidade substancial, gerar variantes resistentes, e são frequentemente ineficazes. Em contraste, as células T-vírus específicos derivados de doadores de células-tronco se mostraram seguros e eficazes para a prevenção e tratamento da infecção viral ou doença no transplante de células-tronco hematopoiéticas (TCTH) definição 2,5,6,18-21. No entanto, a mais ampla implementação de T imunoterapia celular é finalmente limitado pelo custo, complexidade e tempo necessário para a produção de CTL. Nosso romance e uma abordagem rápida para gerar multivirus CTL, descrito no manuscrito atual, deve aumentar substancialmente a possibilidade de terapia com células T citotóxicas para doenças virais, permitindo que a estratégia para se tornar um padrão de atendimento para o hospedeiro imunocomprometido. O uso de plasmídeos DCs nucleofected como APCs permite apresentação de antígenos em ambos MHC classe I e II sem a concorrência de vetores virais ou mesmo de vários antígenos virais sendo expresso em uma única célula desde diferentes populações DC são utilizados para cada três plasmídeo. O uso de IL-4 / 7 aumenta a sobrevida das células T e proliferação, o que ajuda a aumentar correspondentemente a freqüência eo repertório de responder células T antígeno-específicos 16,17. Finalmente, a cultura no G-Rex reduz drasticamente a apoptose de células T durante a cultura. Troca gasosa (O 2 e CO 2) ocorre através de uma membrana de silício de gás permeáveis ​​na base do frasco, evitando a hipóxia, permitindo uma maior profundidade de médio acima das células, fornecendo mais nutrientes e diluição de resíduos. Esta plataforma também pode ser estendido aos vírus adicionais como quando antígenos protetores são identificados.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> CellGenix </td><td> CellGenix </td><td> 2005 </td></tr><tr><td> IL4 </td><td> R + D Systems </td><td> 204-IL/CF </td></tr><tr><td> IL7 </td><td> Peprotech </td><td> 200-15 </td></tr><tr><td> Hyclone RPMI 1640 </td><td> Thermo Scientific </td><td> SH30096.01 </td></tr><tr><td> AB soro humano </td><td> Vale Biomedical Inc. </td><td> HP1022 </td></tr><tr><td> Nucleofector </td><td> Amaxa / Lonza </td><td> AAF-1001B &amp; AAF-1001X </td></tr><tr><td> Kit Nucleofection </td><td> Amaxa / Lonza </td><td> V4XP-3012 </td></tr><tr><td> Plasmídeos </td><td> NTC </td><td> n / a </td></tr><tr><td> GM-CSF </td><td> R &amp; D </td><td> 215-GM/CF </td></tr><tr><td> IL1 </td><td> R &amp; D </td><td> LB-201-025 </td></tr><tr><td> IL6 </td><td> R &amp; D </td><td> 206-IL-CF </td></tr><tr><td> TNFa </td><td> R &amp; D </td><td> 210-TA-010 </td></tr><tr><td> PGE2 </td><td> Sigma </td><td> P6532-1MG </td></tr><tr><td> G-Rex </td><td> Wilson Lobo Manufacturing </td><td> AY11-00027 </td></tr></tbody></table>

generation of multivirus-specific t cells to prevent/treat viral infections after allogeneic hematopoietic stem cell transplant

Infecções virais causar morbidade e mortalidade em transplante de células-tronco hematopoéticas alogênico (TCTH) destinatários. Nós e os outros têm gerado com sucesso e infundida células T específicas para Epstein Barr (EBV), citomegalovírus (CMV) e adenovírus (Adv), utilizando monócitos e células EBV-transformadas linfoblastóides (EBV-LCL) gene modificado com um vetor de adenovírus como células apresentadoras de antígenos (APCs). Sómente 2x10 5 / kg trivirus específicos linfócitos T citotóxicos (CTL) proliferaram por vários registros após a infusão e apareceu para prevenir e tratar a doença viral grave ainda resistentes a outras terapias disponíveis. Quanto mais ampla a implementação desta abordagem é limitada pela encorajando altos custos de produção, a complexidade de produção e tempo prolongado (4-6 semanas para EBV-LCL geração, e 4-8 semanas para a fabricação de CTL - total 10-14 semanas) para a preparação. Para superar essas limitações, temos desenvolvido um novo protocolo de GMP-compliant produção CTL. Em primeiro lugar, no lugar de adenovectors para estimular as células T que usamos células dendríticas (DCs) nucleofected com DNA de codificação de plasmídeos LMP2, EBNA1 e BZLF1 (EBV), hexon e Penton (Adv), e pp65 e IE1 (CMV), como apresentadoras de antígenos células. Estes APCs reativar células T específicas para todos os antígenos estimulante. Cultura, segundo células T ativadas na presença de IL-4 (1000 U / ml) e IL-7 (10ng/ml) aumenta e sustenta o repertório ea freqüência de células T específicas em nossas linhas. Terceiro, temos utilizado um novo dispositivo de cultura de gás permeável (G-Rex), que promove a expansão ea sobrevivência de números de grandes células, após um estímulo único, eliminando assim a necessidade de EBV-LCLS e reduzindo a intervenção do técnico. Ao implementar essas mudanças nós podemos agora produzir CTL multiespecífico segmentação EBV, CMV, e Adv, a um custo por 10 6 células que é reduzida em&gt; 90%, e em apenas 10 dias em vez de 10 semanas, utilizando uma abordagem que pode ser alargado a mais proteção antígenos virais. Nossa abordagem FDA-approved deve ser de valor para aplicações profiláticas e de tratamento para os destinatários de alto risco HSCT alogênico.

Watch this Scientific Journal Video about Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant at JoVE.com

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Um protocolo rápido, simples e de baixo custo para a geração de doador-derivado multivirus específico CTLs (rctl) para infusão de células-tronco hematopoéticas alogênico transplante (TCTH) destinatários em risco de desenvolver infecções CMV, EBV ou Adv. Este processo de fabricação é compatível com GMP e deve garantir a mais ampla implementação de T-cell imunoterapia além de centros especializados.

Geração de células T específicas Multivirus para Prevenir / tratar infecções virais após transplante de células-tronco hematopoéticas alogênico

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

generation-multivirus-specific-t-cells-to-preventtreat-viral

Research

JoVE Journal

Immunology and Infection

17.1K visualizações.  Baylor College of Medicine. Um protocolo rápido, simples e de baixo custo para a geração de doador-derivado multivirus específico CTLs (rctl) para infusão de células-tronco hematopoéticas alogênico transplante (TCTH) destinatários em risco de desenvolver infecções CMV, EBV ou Adv. Este processo de fabricação é compatível com GMP e deve garantir a mais ampla implementação de T-cell imunoterapia além de centros especializados.

Vídeo: Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Seguindo o destino celular, em<em&gt; E. coli</em&gt; Após a infecção por um fago Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Imunologia e Infecção

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 ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Aplicação Clínica de<em&gt; A Bela Adormecida</em&gt; Artificial e Antigen Presenting Cells para modificar geneticamente células T de Sangue do Cordão Umbilical e periférica

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

A identificação de DNA Mutações em Purificada-Tronco Hematopoéticas / células progenitoras

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

O transplante de pele da cauda para estudar as respostas das células T CD4 alogênico em camundongos

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

As células T de enriquecimento de células automatizado de citomegalovírus específico-para aplicações clínicas usando o Sistema de captura de citocinas

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Geração e Multi-fenotípica rastreio de alto teor de<em&gt; Coxiella burnetii</em&gt; mutantes por transposões

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Desenvolvimento de Células-Tronco de derivados de células T regulatórias antígeno específico da Contra Auto-imunidade

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

Medição do T celular aloreactividade usando imagens de Citometria de Fluxo

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Avaliação das respostas de células T Zika vírus específicos em órgãos Immunoprivileged de infectados Ifnar1- / - ratos

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...