Apoiado em parte pelo NIH R01 CA121258, CA125244 R01, R01 CA154256, NIH / NCATS UCLA CTSI Grant Número UL1TR000124, USAMRMC W81XWH-08-1-0734, e do Fundo de Investigação Holmes Memorial Joan S.. MJH e GCO recebeu suporte do Joan S. Holmes Memorial pós-doutorado na Universidade da Califórnia. O dispositivo CSM foi obtido da Creative métodos científicos: www.creative-sci.com. O vector lentiviral foi recebido a partir da UCLA Vector Core, que é suportado por CURA / P30 DK041301.

1. Deslize Preparação <ol><li> Insira os slides Manifold sedimentação celular em bolsas de esterilização e selar com fita crepe. Vire o lado revestido de Teflon da corrediça do lado do papel a da bolsa para evitar depósitos de plástico nos poços. </li><li> Bolsas de autoclave durante 15 min a 121 ° C. </li><li> Remover slide da bolsa de esterilização dentro de uma cabine de segurança biológica e coloque em um estéril 150 x 15 mm estéril placa de Petri. Até quatro lâminas pode caber por prato. Coloque um 35 x 10 mm placa de Petri ao lado dos slides. Adicionar 2-3 ml de H 2 O estéril para proporcionar humidificação. </li><li> Lâminas pré-revestimento com poli-D-lisina a 100 mg / ml usando um volume suficiente para cobrir a totalidade bem. Depois de uma hora, à temperatura ambiente, aspirar a solução e lavar a superfície do poço duas vezes por pipetagem de 1x PBS sobre a superfície dos poços. </li><li> Opcionalmente, adiciona fibronectina ou outros componentes da matriz extracelular para assegurar uma maior aderência das células do tumor. Adicionar fibronectina a 5 jig / ml em volumes suficientes that os poços não seca dentro de um curto período de tempo à temperatura ambiente. <ol><li> Após 1 hora, aspirar a solução de sobra e lavar duas vezes por pipetagem cima e para baixo com 1x PBS. </li></ol></li><li> Manter PBS aos poços até tumor está pronto para a galvanização. Uso desliza no mesmo dia. </li><li> Colheita de um balão confluente do tipo de células de tumor aderentes desejado. Contagem de células viáveis ​​usando corante Trypan Blue exclusão por microscopia de luz e ressuspender em 5 x 10 6 células / ml em meio de crescimento, por exemplo adequadamente tamponada, meio essencial mínimo de Dulbecco (DMEM) com 10% de soro fetal bovino (FBS), L glutamina e piruvato de sódio. </li><li> Se imagiologia de fluorescência da população de células aderentes se desejado, rotular a população de células aderentes com um corante fluorescente ou um corante vital proliferação de células, seguindo os protocolos fornecidos pelo fabricante. </li><li> Suavemente pipeta 10 ul de meio completo em cada cavidade da lâmina, tendo o cuidado de evitar a criação de bolhas na microcavidadels, pois estes podem impedir a adesão de células tumorais uniforme. </li><li> Adicionar 2,5-5,0 x 10 4 células (5-10 ul de suspensão de células) para o meio em cada poço (Figura 1A). Ajustar o número óptimo dependendo do tipo de célula de tumor e o número de dias de crescimento desejados. Células tumorais maiores ou com células em crescimento rápido pode exigir menor quantidade de células inicialmente. Traga bem volume de até 40 mL, adicionando médio e pipeta cima e para baixo para assegurar uma distribuição uniforme das células. </li><li> Depois de todas as cavidades são semeadas, permitir que as células tumorais a aderir à TA, em seguida, colocar a tampa na de 150 x 15 mm numa caixa de Petri e cuidadosamente mover o prato para uma incubadora humidificada a 37 ° C / 5% de CO 2 durante pelo menos 24 horas. </li><li> Alterar o meio de cultura diária. Pipetagem cuidadosa de uma porção do meio gasto a partir da monocamada e substitua com meio completo fresco. NOTA: Por causa da evaporação, mais volume terá de ser adicionado do que foi retirado. Wells estará pronto para o ensaio quando um mesmo, conflefluente camada de células está presente. Este é tipicamente de 1-3 dias após a sementeira inicial. </li><li> Lavar monocamada suavemente uma vez com PBS como descrito no passo 1.4 e prosseguir para a etapa 2, se monocamadas são desejados, ou para o passo 1.13 abaixo para extrair proteínas da MEC da monocamada. </li><li> Adicione uma solução de 0,5% de Triton-X (v / v) em meio completo e adicionar 30 ul a cada poço. Deixe monocamada digerir na capa durante 2 min, em seguida, lavar duas vezes com meio completo por pipetagem como descrito acima. NOTA: A camada de ECM podem ser visíveis por microscopia de luz (Figura 2). Use os slides imediatamente, ou manter-se em meio completo no umidificado incubadora de CO 2 por 1-2 dias. Não permitir que os poços para secar. </li></ol> 2. A sedimentação de células T não aderentes Onto Deslize <ol><li> Se imagiologia de fluorescência da população de células não aderentes for desejado, rotular as populações de células não aderentes com um corante fluorescente vitais, tais como CFSE, Cell Rastreio Vermelho CMPTX, ou eFluor670, seguindo os protocolos fornecidos pelo fabricante, pelo menos, 1 hora antes do ensaio. Alternativamente, manipular as células para expressar proteínas fluorescentes codificados por vectores virais. </li><li> Autoclave o colector de sedimentação de células numa bolsa de autoclave tal como descrito no ponto 1.2 acima. </li><li> Retire a câmara úmida contendo lâminas revestidas de Teflon da incubadora e lugar na cabine de segurança biológica. </li><li> Lave os poços com PBS 1x por pipetagem cima e para baixo, em seguida, adicione 45 mL de meio completo, contendo pelo menos 10% de soro. </li><li> Remover o colector do envelope de esterilização e cuidadosamente deslizar ao longo dos poços até o &#39;gancho&#39; no final do colector toca o fundo da corrediça (Figura 1B). </li><li> Certifique-se de que o meio de cultura é visível em cada um dos canais. Se nenhum for visto, tirar o colector e adicionar mais meio para o correspondente bem, em seguida, substituir e verificar novamente. </li><li> Repita o passo 2,5 para cada channel do colector, ou para o maior número de poços, como desejado. </li><li> Contagem de células não-aderentes e ressuspender em não menos do que 1,0-2,0 x 10 5 / ul. Desenha-se 1 ml de células e pipeta-los lentamente no interior do canal (Figura 1C). Faça isso para cada bem desejar. </li><li> Deixar todo o aparelho carregado de células, isto é, o colector e corrediça, no interior da bio-capuz durante 20-30 minutos para permitir que as células do canal para assentar. </li><li> Remover o colector tocando ambas as extremidades e suavemente levantando-o da lâmina de modo a não perturbar as células semeadas focal no centro das cavidades (Figura 1D). </li><li> Inspeccionar cada poço para assegurar que as células não aderentes foram semeados com sucesso no centro do poço ficar dentro da circunferência do canal colector. As células não aderentes irá aderir idealmente ligeiramente à monocamada ou ECM e uns com os outros, por conseguinte, se mover ligeiramente as lâminas em torno não fará com que a célula Pellet para dispersar. Não disputam o slide. </li></ol> 3. Microscopia de Fluorescência <ol><li> Com um equipamento experimental adequado, realizar a imagem após a sedimentação é confirmado. Idealmente, usar um microscópio que está equipado com uma câmara de CO2 e humidade. Imagem com baixa potência, ou seja, 4X ou 10X, para permitir imagens múltiplas para ser tomado de uma área maior, de modo que a totalidade do bem pode ser visto. </li><li> Aquisição de imagens digitais usando software de captura de imagem. Realizar campo claro e / ou multi-canal de imagens por fluorescência. </li><li> Se desejar, configure um lapso de tempo para gravar imagens em diferentes canais sobre uma determinada área, ou manter o slide umidificado e em uma temperatura de 37 ° C / 5% de CO2 e manualmente tirar a imagem nos momentos desejados (Figura 1E &amp; F, recomendado para pontos de tempo mais longos). </li></ol> 4. Análise e Visualização <ol><li> Usando software de edição de imagem, luz e fl mesclaruorescent imagens em uma camada de modo a vários tipos de células e de marcadores pode ser visto na mesma imagem. Arraste e solte os arquivos de imagem para cada cor individual para o programa e, quando solicitado, selecione a opção &quot;Add Layer&quot; para criar um arquivo de imagem com várias camadas. <ol><li> Na maior ampliação, tome, alinhar e costurar as imagens de todo o bem digitalmente. Alinhar arquivos por olhando para regiões conservadas entre as duas imagens e sobrepondo uma imagem em cima da outra, até que um quadro completo é gerado. </li></ol></li><li> Adicionar bares mícron de imagens durante a aquisição utilizando o software de captura para determinar a distância células individuais têm migrado. </li><li> Para determinar a migração de células em vários momentos, fazer mapas de intensidade de fluorescência superfície com software de imagem, ou seja, ImageJ, para quantificar a agregação de células T fluorescente ou espalhar ao longo do tempo. <ol><li> Para gerar terrenos de superfície, dê uma imagem em camadas gerado na etapa 4.1 e sob a janela Layers,desmarque todas as camadas, exceto aqueles correspondentes ao canal desejado. Por exemplo, se uma parcela de superfície é desejada para visualizar a expressão de EMD, apenas as camadas &quot;verdes&quot; correspondentes ao filtro usado para este marcador deve ser visível. </li><li> Achatar a imagem e salvá-lo em um formato de arquivo reconhecido (por exemplo, .png) e carregar a imagem no programa. Para gerar a plotagem de superfície, altere o tipo de imagem para 8 bits, alterar o brilho / contraste conforme necessário para reduzir o fundo, e selecione a opção &quot;Analisar / Surface Plot &#39;. As imagens na Figura 5 foram geradas, verificando o &quot;Sombra&quot;, &quot;Desenhe Axis &#39;,&#39; One Polygon Per Line &#39;, e caixas de seleção&#39; Smooth &#39;. </li><li> Em alternativa, tomar medidas utilizando o raio ou o diâmetro do depósito celular focal no tempo zero e comparar com as células no ponto mais externo em momentos diferentes. O diâmetro do Teflon poços é de 6,0 mm. </li></ol></li></ol>

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The authors declare they have no competing financial interests.

As células tumorais em uma única suspensão de células foram pipetadas para os poços de uma lâmina de Teflon-mascarado. As células foram deixadas aderir e, em seguida, formado monocamadas numa atmosfera humidificada com 5% de CO2, 37 ° C incubadora (Figura 1A). Monocamadas estabelecidas ou proteínas da MEC derivados de monocamada podiam ser colhidas para estes ensaios (Figura 1B). Linfócitos T efectoras marcadas com corantes fluorescentes vitais ou transduzidas com v...

O ensaio de migração celular radial monocamada foi originalmente desenvolvido para medir as propriedades infiltrativas de células tumorais aderentes 1-4 em lâminas revestidas com matriz extracelular (ECM) 5-7 proteínas ou com componentes de ECM individuais, tais como fibronectina ou laminina 1,2. A técnica de semeadura envolvida uma única suspensão celular de células de tumor no centro de poços, utilizando um colector de células de sedimentação aço inoxidável (CSM). Após a sedimentação, as células tumorais se aderir ao fundo do poço e a mudança no diâmetro da população inicial de células ao longo do tempo foi usada para estabelecer uma taxa de motilidade horizontal. O ensaio de migração celular Radial Monolayer proporcionaram uma vantagem visual sobre outros métodos existentes que empregavam transwell placas de ensaio in vitro os recursos migratórios de células; estes ensaios não são propícias à geração de imagens 8. Como assim, ele também forneceu uma grande quantidade de liberdade na escolha do timepointé quando a migração é avaliada, sem limite do número de pontos no tempo um pesquisador pode optar por imagem após a sedimentação. Porque a capacidade de migrar é uma funcionalidade importante para as células não aderentes, em especial na área da imunoterapia ou onde eles podem ser utilizados como veículos de administração para os vectores virais, nós adaptamos o uso do MCS para avaliar a migração de células não-aderentes tipos sobre monocamadas de células de tumor, além das proteínas de ECM. O benefício adicional de microscopicamente visualizando a migração de células não-aderentes em monocamadas de células tumorais viáveis, em ECM complexo isolado a partir do tumor, ou sobre os componentes da ECM individuais torna este ensaio versátil. Os ensaios que utilizam poços revestidos com uma única proteína extracelular não refletem exatamente o substrato tecido ECM ou tumor das células que migram através in vivo. Aqui, usamos alorreativas linfócitos T citotóxicos (alloCTL), sensibilizados a major histocompcomplexo atibility (MHC) proteínas utilizando reacções unidireccional mista de linfócitos de células de tumor (MLTR) ou reacções de linfócitos mistos (MLR) 9, como o nosso tipo de células não-aderentes representativo. Testamos células de origem humana e murina. Quando a migração foi medida em monocamadas de tumor, as células tumorais utilizadas foram alvos ou parcialmente relevantes, exibindo algumas das mesmas proteínas MHC encontrados na população de células usadas para sensibilizar as efetoras, ou alvos totalmente pertinentes, com um conjunto completo de moléculas MHC que o efetores foram sensibilizados no sentido. Em algumas experiências, foi utilizado CellTracker fluorescente vermelha CMPTX ou corante proliferação celular eFluor 670 para diferenciar entre células efectoras e alvo. Utilizou-se também a transdução com vectores virais para codificação de proteínas fluorescentes como uma forma adicional de visualizar as células. Para certos ensaios, nós transduced o alloCTL com vetores retrovirais replicantes (RRV) que codificam para o verde esmeralda (EMD) proteína fluorescente 10,11; para others, as células tumorais foram transduzidas com vectores lentivirais que codificam para mStrawberry. O alloCTL foram semeadas por meio de um canal do colector para o centro de qualquer monocamadas de células tumorais ou de ECM colhidas a partir de monocamadas de células de tumor. Interacções de células aderentes e não aderentes foram visualizadas por luz e / ou por microscopia de fluorescência ao longo do tempo. Perturbação na monocamada de células tumorais em baixa potência, ou tumorais células com núcleos fragmentados em alta potência foram indicadores de lesão celular por lise e apoptose, respectivamente. Nós digitalmente criados mapas de intensidade fluorescente superfície que mostram a migração de células fluorescentes T não aderentes sobre as culturas em monocamada. Observamos, também, a citotoxicidade engendrado para a monocamada de células de glioma aderente após a formação do cluster do alloCTL não aderente sobreposta. Assim, foi observada a transdução horizontal de RRV-EMD do alloCTL à monocamada de glioma.

<table border="0" cellpadding="0" cellspacing="0" width="920">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Name of Reagent/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:239px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:312px;">Teflon-masked microscope slides</td>
			<td style="width:203px;">Creative Scientific Methods</td>
			<td style="width:239px;">CSM002</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:312px;">Cell Sedimentation Manifold</td>
			<td style="width:203px;">Creative Scientific Methods</td>
			<td style="width:239px;">CSM001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Petri Dish, 150mm</td>
			<td style="width:203px;">Corning</td>
			<td style="width:239px;">430597</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Petri Dish, 35mm</td>
			<td style="width:203px;">Corning</td>
			<td style="width:239px;">430588</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Phosphate-Buffered Saline (PBS)</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">21-040</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">20 &#956;L Pipetman</td>
			<td style="width:203px;">Gilson</td>
			<td style="width:239px;">F123600</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">200 &#956;L Pipetman</td>
			<td style="width:203px;">Gilson</td>
			<td style="width:239px;">F123601</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">200 &#956;L pipette tips</td>
			<td style="width:203px;">VWR</td>
			<td style="width:239px;">89003-060</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Distilled, deionized water (sterile)</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">25-055</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Trypsin</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">25-054-CL</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Celltracker Red CMPTX</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:239px;">C34552</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">TrypLE Express (optional)</td>
			<td style="width:203px;">Gibco</td>
			<td style="width:239px;">12604</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Tumor cell culture media (e.g. DMEM)</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">10-013</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">AIM-V serum-free media</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:239px;">12055-083</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Fetal Bovine Serum</td>
			<td style="width:203px;">Omega Scientific</td>
			<td style="width:239px;">FB-02</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Inverted microscope</td>
			<td style="width:203px;"></td>
			<td style="width:239px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">SPOT Advanced</td>
			<td style="width:203px;">Diagnostic Instruments</td>
			<td style="width:239px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Poly-D-Lysine</td>
			<td style="width:203px;">Millipore</td>
			<td style="width:239px;">A-003-E</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Fibronectin</td>
			<td style="width:203px;">BD</td>
			<td style="width:239px;">354008</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">31/2 x 51/4 Autoclave Pouches</td>
			<td style="width:203px;">Crosstex</td>
			<td style="width:239px;">SCXS2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Trypan Blue</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">25-900-CI</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Cell Proliferation eFluor 670</td>
			<td style="width:203px;">eBioscience</td>
			<td style="width:239px;">65-0840-85</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">ImageJ</td>
			<td style="width:203px;">NIH</td>
			<td style="width:239px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

radial mobility and cytotoxic function of retroviral replicating vector transduced, non-adherent alloresponsive t lymphocytes

Relatamos uma nova adaptação do ensaio de migração celular radial monocamada, relatada pela primeira vez para medir a migração radial das células tumorais aderentes em proteínas de matriz extracelular, para medir a motilidade das células imunitárias fluorescentemente marcado, humano ou não-aderente efectoras de murino. Esta técnica utiliza um colector de aço inoxidável e 10 bem corrediça de Teflon para focalmente depositar as células T não-aderentes preparadas em poços quer com monocamadas confluentes de células de tumor ou proteínas da matriz extracelular. Microscopia de fluorescência de luz e / ou multi-canal é usado para rastrear o movimento eo comportamento das células efetoras ao longo do tempo. Corantes fluorescentes e / ou vectores virais que codificam transgenes fluorescentes são usados ​​para marcar diferencialmente os tipos de células para a imagiologia. Este método é distinta da de tipo semelhante em ensaios in vitro que rastreiam migração horizontal ou vertical / invasão utilizando câmaras de slides, agar ou transwell placas. O ensaio permite que os dados detalhados de imagem para be coletados com diferentes tipos de células que se distinguem por marcadores fluorescentes específicos; mesmo subpopulações específicas de células (isto é, transduzidas / nontransduced) pode ser monitorizado. Superfície parcelas intensidade de fluorescência são gerados usando canais de fluorescência específicas que correspondem ao tipo de célula de migração. Isto permite uma melhor visualização da mobilidade celular imune não-aderente em momentos específicos. É possível a obtenção de provas de outras funções das células efectoras, tais como citotoxicidade ou a transferência de vectores virais a partir de células de efector para alvo, bem como. Assim, o método permite que os investigadores microscopicamente documentar célula-a-célula interacções de diferencialmente marcado, não-aderente com células aderentes de vários tipos. Tais informações podem ser especialmente relevante na avaliação dos tipos de células do sistema imunológico biologicamente manipulados ou ativados, onde a prova visual de funcionalidade é desejados com células-alvo tumor antes de sua utilização para a terapia do câncer.

Os vectores virais que codificam para as proteínas fluorescentes pode ser utilizada em adição a, ou em vez de, corante fluorescente. Transdução virai deve ser executada antes do ensaio de motilidade. Ambos os tipos de células aderentes e não aderentes podem ser diferencialmente marcado. O protocolo para a transdução vai depender do tipo de vector utilizado. Aqui, nós transduced o alloCTL nas Figuras 3, 5 e 6 com RRV-EMD, pelo menos, dois dias antes do ensaio utilizando o proto...

Watch this Scientific Journal Video about Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes at JoVE.com

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

Radial Mobilidade e função citotóxica de Retroviral Replicando Vector transduzidas, Alloresponsive não aderentes Linfócitos T

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...

radial-mobility-cytotoxic-function-retroviral-replicating-vector

Research

JoVE Journal

Immunology and Infection

7.7K visualizações.  UCLA David Geffen School of Medicine. We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality. 

Vídeo: Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

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

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 presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

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 ...

Imunologia e Infecção

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

Preparação e Uso de HIV-1 infectados CD4 + T-Primária células como as células-alvo em Ensaios Natural Killer Celular citotóxicos

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

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

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

Métodos para avaliar Mediated Beta Morte celular por linfócitos T citotóxicos

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

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

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

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

Expandindo linfócitos T citotóxicos do sangue do cordão umbilical que citomegalovírus Target, vírus de Epstein-Barr, e Adenovirus

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

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Diferenciação de células Dirigido tronco pluripotentes induzidas no sentido de Linfócitos T

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

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

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

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

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

Isolamento e Th17 Diferenciação de Linfócitos T CD4 Naïve

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

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

Estática Adesão Ensaio para o Estudo da integrina Ativação em Linfócitos T

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Protocolo de isolamento eficiente para linfócitos B e T a partir de tonsilas palatinas Humanos

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

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, ...