Christian Welker is funded by a grant provided by the J&#252;rgen-Manchot-Stiftung.

Declaração de ética: Todos os procedimentos foram realizados de acordo com a Declaração de Helsinki e foi aprovado pelo Comitê de Ética da Clínica da Universidade de Tübingen (projetos 38 / 2009B02 e 470 / 2013B02). 1. Isolamento de Células Mononucleares de Sangue Periférico (PBMC) <ol><li> Tome 50-100 ml de um voluntário saudável por meio de venipunctura usando uma seringa de 50 ml contendo 1000 UI de heparina e sulfato de diluir o sangue 1: 2 com PBS (pH = 7,2). </li><li> Camada cuidadosamente 35 ml de sangue em solução de PBS: solução de separação de 15 ml de sangue, tais como Biocoll (d = 1,077 g / ml) num tubo de 50 ml. Centrifugar durante 15 minutos a 800 xg à temperatura ambiente. NOTA: O freio não deve ser utilizado. </li><li> Recolher as células da camada linfocítica com uma pipeta e lava-se as células duas vezes em, pelo menos, 50 ml de PBS (centrifuga-se durante 12 min; a 400 xg). Remover o sobrenadante após centrifugação com uma pipeta. </li><li> Lyse restantes eritrócitos por suspensão e incubAting a fracção linfocítica com 1-5 ml de uma solução tampão hipotónico para 2-4 min. NOTA: A duração da exposição e do volume de tampão de lise dependem do tampão de lise utilizada e a quantidade de glóbulos vermelhos restantes na fracção de linfócitos. </li><li> Dilui-se as células com PBS (1:10) e centrifugar a 250 xg durante 12 min e, subsequentemente, lavar e ressuspender o sedimento de células uma vez em 10 ml de PBS a 300 xg durante 12 min. </li><li> Ressuspender os linfócitos em PBS e contar as células numa câmara de contagem Neubauer usando solução azul de Tripano. NOTA: Normalmente,&gt; 1 x 10 8 a partir de linfócitos são recebidos 100 ml recentemente tirado sangue periférico. </li></ol> 2. Isolamento de Células T Vδ1 <ol><li> Take &lt;1 x 10 6 linfócitos isolados por um período inicial de FACS que determinam a frequência de células T CD4 Vδ1. NOTA: O tamanho da população desta entidade de células T podem diferir muito entre os indivíduos e de acordo com seu estado imunológico. Neossoloaliado, cerca de 1% das células T são Vδ1 + e cerca de 1-5% de células T CD4 + são Vδ1 +. <ol><li> Dilui-se as células com 1 ml de FACS-tampão, que consiste em PBS contendo FCS a 2% e EDTA 5 mM. Centrifugar a 660 xg durante 2 minutos e decantar o sobrenadante. O volume de tampão de FACS de refluxo cerca de 100 ul é suficiente para a coloração subsequente. Resumidamente vórtex e incubar as células com 5 ul de reagente de Fc-bloqueio durante 5 min à TA, durante o aumento da especificidade da coloração FACS. </li><li> Adicionar anticorpos monoclonais humanos directamente para as células sem remover o reagente de bloqueio de Fc. Certifique-se de corar as células com anticorpos contra Vδ1 (1:50), CD3 (1:50), CD4 (1: 200), CD8 (1: 200) e TCRαβ (01:25). Prepara-se o controlo do isotipo de imunoglobulina com as correspondentes isotipos. Incubar as células com anticorpos durante 20 min a 4 ° C no escuro. </li><li> Lave as células em 1 ml de tampão FACS (centrifugar por 2 min; a 660 xg), decantar o supernatant e proceder à aquisição FACS no volume tampão restante. </li></ol></li><li> Peletizar as células (que não são utilizadas na análise de FACS) por centrifugação durante 12 min; a 300 g; 8 ° C. Remover o sobrenadante e ressuspender a célula pelletin 60 ul MACS-tampão por 1 x 10 7 células. Adiciona-se 20 de Reagente de Bloqueio-Fc? L por 1 x 10 7 células e incubar durante 5 min a 4 ° C. </li><li> Adicionar 10 ul de anticorpo anti-humano Vδ1 conjugado com FITC por 1 x 10 7 células directamente para as células. Incubar durante 12 min a 4 ° C no escuro. </li><li> Lavam-se as células utilizando 14 mL de tampão-MACS (centrifuga-se durante 12 min; a 300 g; 8 ° C). Elimine o sobrenadante completamente e voltar a suspender as células em MACS tampão 80 ul por 1x10 7 células. </li><li> Tome 1-2 x 10 5 células e diluí-los em tampão FACS 100 ul para verificação da rotulagem por análise FACS. Não são necessários outros aditivos para este passo. Certifique-se de que existe um suficient diferença no brilho para Vδ1 - e células Vδ1 +. Se este não for o caso, repetir os passos 2.3) e 2.4). </li><li> Adicionar 20 pi microesferas anti-FITC por 1 x 10 7 células para as células restantes, misturar bem e incubar durante 15 min no escuro a 4 ° C. Em seguida, lavar as células utilizando MACS ml de tampão de pelo menos 10 (centrifuga-se durante 12 min, a 300 xg; 8 ° C). </li><li> Durante a centrifugação, equilibrar a coluna magnética pré-arrefecida em um íman colocado com 500 ul de tampão MACS. </li><li> Ressuspender as células em 500 ul MACS-tampão (para o número de células maior do que 1 x 10 8, adaptar-se de acordo com este volume) e aplicam-se as células cuidadosamente na coluna. Recolher o flow-through contendo a Vδ1 - células. </li><li> Lava-se a coluna três vezes com 500 ul de tampão-MACS e recolher o fluxo de passagem de novo no mesmo tubo. Note-se que o reservatório tem de estar vazia antes da aplicação na coluna de tampão. </li><li> Remova a coluna do dispositivo magnético e colocá-lo em um tubo cônico de 15 ml. Rapidamente lavar a coluna com tampão de 1 ml por MACS firmemente empurrando o êmbolo para a coluna. Recolher o eluído num tubo. NOTA: De modo a obter um grau de pureza&gt; 98%, é geralmente necessário repetir os passos de 2,7-2,9) com uma segunda coluna. </li><li> Verifique a pureza das células por análise de FACS 18 com o mesmo painel de anticorpos, como anteriormente (ver 2.1.2) e contar as células. </li></ol> 3. Isolamento de Células T + Vδ1CD4 <ol><li> Ressuspender 25 ul de pérolas de CD4 com um vortex para&gt; 30 seg. Lavar as pérolas (a quantidade mínima como descrito pelo fabricante) em 1 ml de tampão de MACS em um tubo de 1,5 ml de reacção, colocando o tubo num íman durante 1 min. Descartar o sobrenadante cuidadosamente com uma pipeta de pequena escala (200 mL) e ressuspender as esferas no volume original de tampão-MACS. </li><li> Pelotizar Vδ1 + células (centrifugar por 12 min; a 300 xg) e aspirar o sobrenadante e ressuspender as células todas Vδ1 + em 500 ul de tampão-MACS e adicioná-los para o tubo contendo os grânulos. Misturar vigorosamente e incubar durante 20 min a 4 ° C com inclinação constante (por exemplo, em um rotor). </li><li> Colocar o tubo contendo as células em um magnete durante 2 min. Certifique-se de que não existem vestígios na tampa. NOTA: As células T CD4 + terá ligado a esferas magnéticas e anexar ao lado do tubo virado para o íman. </li><li> Abra cuidadosamente a tampa, mantendo o tubo no interior do dispositivo magnético e recolher o sobrenadante que contém o CD4 - células Vδ1 + utilizando uma pipeta de pequena escala. Coloque - CD4 + Vδ1 células para um tubo separado e colocar este tubo para o íman de novo para evitar possivelmente remanescente células CD4 ou grânulos a partir da população. <ol><li> Lava-se a CD4 - células em 5 ml de tampão-MACS (centrifuga-se durante 12 min; a 300 xg) umND ressuspender-los em uma concentração de 1 x 10 5 células por 100 uL meios. - Células CD4 pode ser facilmente cultivado sob as condições dadas abaixo (4.2). </li></ol></li><li> Colocar o tubo com os alvos de células CD4 + fora do campo magnético, ressuspender as células em 500 ul de tampão-MACS e colocá-los de volta para dentro do dispositivo magnético. Repita duas vezes os passos de 3,3-3,5 para obter uma pureza mais elevada. Remover o sobrenadante por pipetagem </li><li> Ressuspender as células CD4 + em meio de cultura 100 ul (RPMI 1640, 10% de FCS, 1% de L-glutamina, 1% de penicilina / estreptomicina) e adicionar 10 ul de uma solução destacando-grânulo. Incubar à temperatura ambiente durante 45 min com inclinação constante (por exemplo, em um rotor). </li><li> Colocar as células em um íman durante 1 min. Recolher cuidadosamente o sobrenadante contendo Vδ1 células CD4 + livre-talão com uma pipeta de pequena escala. </li><li> Fora do ímã, voltar a suspender as contas com meios de cultura de 100 ul e repitaos passos 3.6 e 3.7 por duas vezes para se obter números maiores de células de células CD4 +. </li><li> Peletizar as células (centrifugar a 300 xg, durante 12 minutos) e descartar o sobrenadante completamente por pipetagem. Ressuspender as células em meios frescos, pré-aquecido e contá-los. Examinar a pureza das células CD4 + Vδ1 por análise FACS descritos nos passos 2.1.1-2.1.3. </li></ol> A clonagem de células 4. Único por diluição limitada <ol><li> Isolar células mononucleares do sangue periférico (PBMC) de um dador alogénico como representado na 1,1-1,6. </li><li> Irradiar 2,5 x 10 7 PBMC alogénicas com 80 Gy em 25 ml de meio de cultura utilizando-γ radiação. </li><li> Adicionar IL-2 (200 U / ml), IL-7 (20 ng / ml) e PHA (2 ug / ml) para as células de alimentação irradiadas e distribuí-los em placas de 96 poços formato U, 5 x 10 4 alimentador células em 50 ul por poço. </li><li> Diluir as células Vδ1 CD4 + a uma concentração de 0,3 células por 50 ul. Pipette 50 ul de solução de células em cada cavidade que abriga as células irradiadas e citocinas. NOTA: Assim, as citocinas são diluídas para a concentração final de 100 U / ml de IL-2, 10 ng / ml de IL-7 e 1 ug / ml de PHA. Incubar as placas de 96 cavidades em uma incubadora a 37 ° C, 5% de CO 2 atmosfera humidificada. <ol><li> Opcionalmente, permanecendo cultivar células purificadas Vδ1 + CD4 + como a cultura em massa sob as mesmas condições de cultura como os clones. </li></ol></li><li> Fornecer as células a cada 3-4 dias com citocinas e meios frescos. Para isso, a troca de metade do meio nos poços com 50 ul de meio fresco por pipetagem. Adicionar concentração duas vezes superior de citoquinas para cada suplementação. Adicionar 5x10 4 células de alimentação irradiadas por poço qualquer outra rodada de alimentação. </li><li> Utilizando um microscópio, observar primeiros clones tornam-se visíveis após 3-4 semanas, como eles metabolizam e, assim, alterar a cor das colónias de meio de cultura e forma. NOTA: Para pelesterap cultivo, ressuspender os clones bem e transferi-los para separar placas de 96 poços. Analisar as células via FACS, conforme indicado na 2.1.1-2.1.3. Clones mantidos nestas condições poderá vir a alterar a sua TCR a partir Vδ1 para αβTCR. O ponto de tempo para esta transdiferenciação varia de clone para clonar. </li></ol>

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The authors have nothing to disclose.

Para estudar o fenótipo, biologia e função de uma entidade celular escassos (T-), nomeadamente células Vδ1 + T CD4 +, foram utilizados dois marcadores: Vδ1 e CD4 por seu isolamento de células magnético positivo. Vδ1 é um receptor órfão, enquanto que CD4 é expresso em células T auxiliares, a um nível inferior em monócitos e células dendríticas, e a um nível muito baixo em células progenitoras hematopoiéticas. As técnicas para o enriquecimento e a selecção de...

Nos vertebrados, a imunidade adaptativa, que está estruturado na celular e uma parte humoral da imunidade desempenha um papel importante na defesa contra agentes patogénicos. O reconhecimento de uma vasta gama de antigénios é mediada por T-hyperpolymorphic e receptores de células B (TCR / BCR), que diz respeito às células T são assumidos para ser produzida principalmente no timo 1. A esta, as células estaminais hematopoiéticas (HSCs), derivadas de medula óssea, o timo sementes e se diferenciam ao longo de etapas bem definidas, finalmente, que dão origem a todas as linhagens de células T. Timo progenitores de semeadura são CD4 - e CD8 - e constituem, assim, a fração de timócitos imaturos, dupla negativa (DN). Sinais derivados do timo, em seguida, induzir a sua linhagem compromisso e a diferenciação em células T, quer ap ou γδ. A expressão de TCR e TCR-y-ô genes das cadeias funcionalmente rearranjados em DN2 / 3 timócitos leva a y δTCR complexos, que levam uma proliferação celulard promover a diferenciação em células γ AT 2,3. Em contraste, o rearranjo de uma cadeia de TCR-β funcional, que pode emparelhar com preTα para construir um preTCR Pt, induz o silenciamento transcricional da cadeia TCR-γ em timócitos DN3 e a sua passagem em células CD4 + CD8 + duplo-positivos timócitos 4 . Nesta fase, a recombinação da cadeia TCR-α ocorre, a exclusão do locus TCR-δ que se aninha no interior do locus TCR-α, revoga assim a produção de um γδTCR nestas células irrevogavelmente 5-9. ΑβTCRs reorganizados são posteriormente seleccionados pela sua capacidade de se ligar a auto-MHC fracamente (seleção positiva), que não pode exceder um determinado limiar para evitar a auto-imunidade (seleção negativa). De acordo com a sua capacidade de se ligar a MHC de classe I ou II, as células seleccionadas αβT desenvolvem em células de um único positivo CD4 + ou T CD8 +, que a saída do timo células T como naive. No entanto, a involução do timo começa cedo na vida levando a exponencialmente reduziu a produção de células T naive que é quase extinto pós-adolescência 10. No entanto, o tamanho do conjunto de células T permanece constante durante toda a vida, o que pode ser explicado apenas parcialmente por uma proliferação homeostática pós-timo de células T e a proliferação de memória imunológica de longa duração 11. Por conseguinte, o desenvolvimento de células T extrathymic deve ocorrer. Uma pesquisa recente ganhou atração substancial que caracterizou progenitores de células, que αβT-at-extrathymic locais deram origem às células T ap funcional 12-17. No entanto, o conhecimento detalhado sobre precursores de células extrathymic αβT que independente de um timo diferenciam em células αβT é tão fragmentado como o fundo que nós temos na rota tomam assim. Recentemente, identificou a entidade pequeno de células T de Vδ1 + </sup&gt; células T CD4 + como uma αβT γδT prognitor extrathymic célula 18, a qual, quando isolados a partir de sangue periférico de dadores humanos saudáveis, podem transdiferenciar em células αβT num ambiente inflamatório leve. Curiosamente e ao contrário da proliferação homeostática de células T pós-timo, transdiferenciação de células Vδ1 CD4 + gera novos receptores de células T, ampliando assim a diversidade repertório, de modo que, potencialmente, novos antígenos podem ser reconhecidas e podem protecção do hospedeiro contra patógenos recém-adquiridas. Isso aumenta a plasticidade das células T e adiciona um novo caminho até agora pouco apreciado para o desenvolvimento de células T extrathymic. O isolamento quantitativo a partir de fontes linfocítica, a geração de clones de uma única célula e sua expansão eficiente são essenciais para o objectivo de identificar os marcadores e moléculas que desencadeiam essa célula αβT precursor`s desenvolvimento extrathymic.

<table border="0" cellpadding="0" cellspacing="0" width="789">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biocoll Solution</td>
			<td style="width:93px;">Biochrom</td>
			<td style="width:119px;">L 6113</td>
			<td style="width:361px;">lymphocyte separating solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lysing Buffer</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>555899</td>
			<td>lysis of erythrocytes&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate-buffered Saline</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td style="width:119px;">D8537&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MACS buffer</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td>supplement with BSA and pre-cool before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BSA</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td>not mandatorily from this supplier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human Vd1 FITC (clone:&#160; TS8.2)</td>
			<td style="width:93px;">Thermo Scientific</td>
			<td>TCR2730</td>
			<td>not mandatorily from this supplier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD3 PerCP (clone: SK7)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>345766</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human TCRab PE (clone: T10B9.1A-31)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>555548</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD4 VioBlue (clone: M-T466)&#160;</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-097-333</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD8 APC-H7 (clone: SK1)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>641400</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Anti-FITC MultiSort Kit</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-058-701</td>
			<td>yields better results than anti-FITC MicroBeads</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MS columns</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-201</td>
			<td>pre-cool before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MiniMACS Separator</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CD4 Positive Isolation Kit</td>
			<td style="width:93px;">life technologies</td>
			<td>11331D</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and ex vivo culture of v&#948;1+cd4+&#947;&#948; t cells, an extrathymic &#945;&#946;t-cell progenitor

O timo, o órgão primário para a geração de células T e ap espinha dorsal do sistema imune adaptativa em vertebrados, tem sido considerada como a única fonte de células αβT. No entanto, a involução do timo começa no início da vida que conduz a uma saída drasticamente reduzido de células αβT naive para a periferia. No entanto, mesmo centenários pode construir a imunidade contra patógenos recém-adquiridas. Uma pesquisa recente sugere o desenvolvimento de células αβT extrathymic, no entanto a nossa compreensão das vias que podem compensar a perda da função do timo são ainda rudimentar. γδ células T são os linfócitos inatos que constituem o principal subconjunto de células T nos tecidos. Recentemente atribuída uma função notável, até agora não apreciado a um subconjunto de células T γδ, mostrando que a entidade escasso de células CD4 + Vδ1 + γδ células T podem transdiferenciar em células αβT em condições inflamatórias. Aqui, nós provide o protocolo para o isolamento do presente progenitoras do sangue periférico e o seu cultivo subsequente. Vδ1 células são enriquecidas a partir de PBMC positivamente de dadores humanos saudáveis ​​usando esferas magnéticas, seguido por um segundo passo em que temos como alvo a fracção escasso de células CD4 + com uma outra técnica de marcação magnética. A força magnética do segundo rótulo for superior a da primeira etiqueta magnética, e assim permite o isolamento positivo eficiente, quantitativa e específico da população de interesse. Em seguida, introduzir o estado da técnica e da cultura requerido para a clonagem eficiente e expansão das células e para a identificação dos clones gerados por análise de FACS. Assim, nós fornecemos um protocolo detalhado para a purificação, a cultura e expansão ex vivo de células T γδ CD4 + Vδ1 +. Este conhecimento é pré-requisito para estudos relacionados a esse αβT progenitor`s celulares biologia e para aqueles que pretendem identify os gatilhos moleculares que estão envolvidos na sua transdiferenciação.

A Figura 1 ilustra as diferentes fases e o resultado do isolamento das células T Vδ1 do sangue periférico. Figura 1A mostra uma distribuição típica das células Vδ1 + em linfócitos CD3 +, bem como a expressão do co-receptor da população Vδ1 +. Neste dador, a frequência de células Vδ1 + (vermelho) é de 2,3% das contagens totais de linfócitos e a expressão de CD4 (verde) de linfócitos Vδ1 + é de 2,6%. No total, a ...

Watch this Scientific Journal Video about Isolation and Ex Vivo Culture of VÎ´1+CD4+Î³Î´ T Cells, an Extrathymic Î±Î²T-cell Progenitor at JoVE.com

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

Isolamento e<em&gt; Ex Vivo</em&gt; Cultura de Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; Células T, γδ um Extrathymic αβT de células progenitoras

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

isolation-ex-vivo-culture-v1-cd4-t-cells-an-extrathymic-t-cell

Research

JoVE Journal

Immunology and Infection

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

9.2K visualizações.  Children's University Hospital Tübingen. Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral Vδ1+CD4+ T cells that is, as we showed recently, an extrathymic αβ T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

Vídeo: Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Isolamento T de linfócitos humanos, Cultura e Análise da Migração In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Imunologia e Infecção

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Expansão ex vivo de células tumorais T-reativa por Meios de 1/Ionomycin Bryostatin eo Common Gamma Cadeia Formulação Citocinas

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Ex vivo de imagens de células T em fatias Node murino linfáticos com Widefield e microscópios confocal

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

Isolation of Double Negative &amp;#945;&amp;#946; T Cells from the Kidney

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

Isolamento de duplas Células αβ T negativa do Rim

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement 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 ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Um<em&gt; Ex vivo</em&gt; Modelo de um Ataque de T-Cell dirigiu-Oligodendrocyte em Agudos fatias de cérebro

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Isolamento de linfócitos da pele humana para análise fenotípica e<em&gt; Ex Vivo</em&gt; Cultura de Células

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

o<em&gt; Ex Vivo</em&gt; Cultura e Reconhecimento de Padrões Receptor Stimulation of Mouse Intestinal Organóides

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

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 ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Um modelo de cultura primária de células bolsas serosas Ex Vivo frango para estudar a patogênese de doenças infecciosas bolsas serosas vírus

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...