D.E. and M.B.D. are members of a multidisciplinary research platform (MRP) of Ghent University, and the Ghent Researchers on Unfolded Proteins in Inflammatory Disease (GROUP-ID) consortium.

Neste estudo, foram utilizados para adultos (6-8 semanas) fêmeas C57BL / 6. Os ratos foram alojados e produzido de acordo com as diretrizes do biotério da Universidade de Ghent. Todos os procedimentos com animais foram aprovados pelo Comitê de Ética e Cuidado Animal Institucional. 1. Preparação de células mononucleares (MNC) a partir de baço de murganho <ol><li> Sacrifique o rato por deslocamento cervical. </li><li> Posicione o mouse de volta para baixo na placa de dissecação e corrigir o traseiras e dianteiras com alfinetes. </li><li> Esterilizar a-superfície da pele com 70% (vol / vol) de etanol / H 2 O. </li><li> Corte a pele ao longo da linha média do abdome para o tórax e expor o baço utilizando instrumentos cirúrgicos estéreis. </li><li> Após o corte dos tecidos gordos circundantes, colocar o baço em uma placa de 24 poços contendo 1 ml de solução salina tamponada 1x com fosfato (PBS) à temperatura ambiente. </li><li> Dissecar do baço utilizando um escalpelo estéril e transferir as peças para um filtro de nylon de 70 um colocado dentro de uma tina 50 mle. </li><li> Usando 2 ml de um êmbolo da seringa, triturar suavemente os pedaços de baço através do filtro de células e lava-se com 5 ml de PBS 1x. </li><li> Adicionar 3 ml de Ficoll-Paque para um tubo de 15 ml e sobrepor a forma do gradiente de densidade com 5 ml de suspensão celular. </li><li> Centrifugar a 400 xg durante 20 min à temperatura ambiente sem travão. </li><li> Transferir a interfase para um novo tubo de 15 ml, adicionar 10 ml de PBS 1x frio e centrifugar a 300 xg durante 10 min a 4 ° C. </li><li> Ressuspender as células em 2 ml de PBS 1x contendo 0,5% de albumina de soro bovino (BSA) e 1 mM de ácido etilenodiaminotetracético (EDTA) (tampão FACS). Transferir 10 uL da suspensão de células para um pequeno tubo, misturar com 10 ul de 0,4% </li></ol> 2. Enriquecimento, detecção e purificação de células do rato iNKT <ol><li> Enriquecimento de rato splenic linfócitos CD5positive (CD5 +). <ol><li> (Opcional) Mancha 10 5 células com FITC-conjugado anti-CD5 (4 ul de diluição 1:30 / 10 6 células)durante 30 min a 4 ° C no escuro, lava-se com 200 ul de tampão de FACS e ressuspender em 500 ul de tampão de FACS. Preparar uma solução de trabalho a 20 uM de 4 &#39;, 6-diamidino-2-fenilindol (DAPI) em PBS 1x, adicionar 1 mL da mesma a 10 5 células em tampão de SCAF e incubar à TA durante 5 min. Adquirir 10 4 células pré-enriquecido no citómetro de fluxo. <ol><li> Usando FCS-H e FCS-W, porta eletronicamente em células lo FSC-W para excluir dupletos celulares e agregados (Figura 1A). Então eletronicamente portão fora DAPI + (células mortas) dentro do W-lo FSC população (Figura 1B), e usar SSC-A e FCS-A para selecionar eletronicamente a população de linfócitos pelo tamanho (Figura 1C). </li><li> Usando células, dentro da porta de linfócitos, traçar SSC-A versus CD5 e electronicamente portão CD5 + linfócitos, como mostrado na Figura 1D. Adquirir o restante da amostra de pré-enriquecido no citómetro de fluxo. </li&gt; </ol></li><li> Ajustar as contagens de células para 10 7 culas por 90 uL em tampão de SCAF e adicionar 10 ul de microesferas anti-CD5. </li><li> Incubar a 4 ° C durante 15 minutos, lavar uma vez com 4 mL de tampão de FACS e ressuspender em 500 ul de tampão de FACS frio. Enriqueça para CD5 + linfócitos usando separação de células magnéticas (MACS) colunas de acordo com as recomendações do fabricante. </li><li> Opcional) Stain 10 5 células com anti-CD5 FITC-conjugado (4 ul de diluição de 1:30 / 10 6 células) e DAPI como em 2.1.1. Adquirir 10 4 linfócitos enriquecidos no citômetro de fluxo e verifique se as portas eletrônicas criado em 2.1.1. selecione CD5 + linfócitos na fracção celular enriquecido. Adquirir 10 5 células no citómetro de fluxo e análise da percentagem de linfócitos CD5 + presentes na fracção enriquecida tal como mostrado na Figura 1E - H. </li></ol></li><li> Detecção e isolamento de rato iNKT cells por FACS. <ol><li> Ressuspender linfócitos enriquecidos CD5 + a uma concentração de aproximadamente 10 6 células por 20 uL em tampão de SCAF contendo anti-CD16 / CD32 (4 ul de diluição 1:50 / 10 6 células) e αGalCer conjugado com PE / CD1d tetrâmero (1 ug / 10 6 células). </li><li> Incubar durante 30 min à temperatura ambiente no escuro. </li><li> Dilui-se anticorpos de ratinho (mAcs) anti-CD3ε V500 (4 ul de diluição de 1:20 / 10 6 células), anti-CD19 E450 (4 ul de diluição 1:50 / 10 6 células) e anti-CD8a E450 (4 ul de diluição 1:50 / 10 6 células) em tampão de SCAF, adicionar aos linfócitos CD5 + e incubar durante 30 min a 4 ° C no escuro. </li><li> Lavam-se as células com 1 ml de tampão de FACS por centrifugação a 300 xg durante 10 min a 4 ° C e ressuspender em 4 ml de tampão de FACS a uma concentração final de 10 7 células / ml. </li><li> Filtrar as células através de um coador de células de 30 ^ m e colocar as células em gelo. </li><li> Calibrar o citômetro de fluxo para separação de células de acordo com as recomendações do fabricante 14,15. NOTA: A calibração de um citómetro de fluxo para separação de células requer treino e pode ser realizada por um operador experiente, ou um técnico treinado. </li><li> Adicionar 10 ul de uma solução de trabalho a 20 ^ M de DAPI por 10 6 células e incubar a temperatura ambiente durante 5 min. Adquirir aproximadamente 10 4 enriquecidos + linfócitos CD5 no citômetro de fluxo. Eletronicamente portão em células lo FSC-W para excluir dupletos celulares e agregados (Figura 2A). Então eletronicamente portão fora DAPI + CD8 + CD19 + células (canal dump) dentro do FSC-lo W população (Figura 2B), e usá-SSC-A e FCS-A para selecionar eletronicamente linfócitos (Figura 2C), como descrito em 2.1.1.1 . <ol><li> Plot αGalCer / CD1d tetrâmero por CD3ε e eletronicamente portão αGalCer / CD1dcélulas tetrâmero + + CD3ε iNKT como mostrado na Figura 2D. Adquirir mais de 2 x 10 5 CD5 + linfócitos enriquecidos para determinar a percentagem αGalCer / CD1d tetrâmero + + CD3ε iTNK células presentes na fracção celular enriquecido. </li></ol></li><li> Utilizando as portas electrónicas criadas em 2.2.7., Células de tipo FACS αGalCer / CD1d tetrâmero + + iNKT CD3ε no citómetro de fluxo, a 70 psi, utilizando um bocal de 70 um, com um caudal de &#39;1,5&#39;. Recolha iNKT células ordenadas em um tubo FACS contendo 1 ml de 100% FCS. Subsequentemente enxaguar células iNKT classificadas a partir do lado do tubo de FACS utilizando 10 ml de RPMI 1640 e centrifugar as células a 300 xg durante 10 min a 4 ° C. </li><li> Ressuspender as células iNKT classificados em 1 ml de RPMI 1640 e 40 ul do mesmo adquirir num citómetro de fluxo para avaliar a pureza das células iNKT ordenadas. Selecione FSC-W lo DAPI - linfócitoss como descrito em 2.2.7. (Figura 2E - G) e electronicamente portão αGalCer / células tetrâmero + + iNKT CD3ε CD1d, como mostrado na Figura 2H. </li></ol></li></ol> 3. Cultura e Expansão do mouse iNKT Cells Dia 0 <ol><li> Revestimento uma placa de 96 poços de fundo plano com anticorpo anti-CD3ε purificados (3 ug / ml) por poço durante 1 h à TA. Lavar duas vezes com 200 ul de PBS 1x e uma vez com 200 ul de meio completo RPMI 1640 (10% vol / vol de FCS, 100 U / ml de penicilina-estreptomicina, 5,5 uM β-mercaptoetanol). </li><li> Contar o número de células viáveis ​​ordenadas iNKT (passo 2.2.8), utilizando um hemocitómetro 0,4% como em 1.11., Centrifugar a 300 xg durante 10 min a 4 ° C e ressuspender em 5 x 10 5 células / ml em meio completo RPMI 1640 contendo IL-2 recombinante de murino (10 ng / ml), recombinante IL-12 murina (1 ng / ml) e solúvel de CD28 anti-rato (5 ug / mL). Placasorted células iNKT 5 a 10 células / poço em placas revestidas com anti-CD3ε e incubar a 37 ° C em uma incubadora de CO 2 durante 2 dias. </li></ol> Dia 2 <ol start="3"><li> Transferir as células para um não revestido de fundo plano de placa de 96 poços de cultura fresco e as células sob condições de repouso, em meio completo RPMI 1640 a 37 ° C em uma incubadora de CO 2 durante 2 dias. </li></ol> Dia 4 <ol start="4"><li> Desalojar as células da placa por pipetagem suave e transferência das células para um novo tubo de 15 ml. Centrifuga-se a 300 xg durante 10 min a 4 ° C, ressuspender em 5 x 10 5 células / ml em meio completo RPMI 1640 contendo IL-7 recombinante de murino (5 ng / ml), e a placa 10 5 células / poço numa plano- placa de 96 poços de fundo durante 4 dias a 37 ° C em uma incubadora de CO 2. </li></ol> Dia 8 <ol start="5"><li> Recolher as células num tubo fresco, centrifugar a 300 xg durante 10 min a 4 ° C e ressuspender em 5 x 10 5 culas / ml em meio completo RPMI 1640 contendo anti-CD28 solúvel (5 ug / mL). </li><li> Revestimento uma placa de 96 poços de fundo plano com anticorpo anti-CD3ε purificados (3 ug / ml) por poço e lavar como em 3.1. Placa 10 5 células / poço em anti-CD3ε placas revestidas e incubar a 37 ° C em uma incubadora de CO 2 até o dia 11. </li></ol> Dia 11-18 <ol start="7"><li> Repita os passos 3,4-3,6. </li></ol> Dia 19-20 <ol start="8"><li> Transferir as células para um novo tubo de 15 ml, centrifugar a 300 x g e ressuspender em 5 x 10 5 culas / ml em meio completo RPMI 1640. </li><li> As células em placa a 10 5 células / poço numa placa de 96 poços de fundo plano e permitir que as células em repouso durante 2 dias a 37 ° C em uma incubadora de CO 2 antes da sua utilização num ensaio. </li></ol> 4. produção de citocinas por células de rato iNKT Usando um CD1d PlatEnsaio de e-bound <ol><li> Revestimento uma placa de 96 poços de fundo plano com 100 uL de murino recombinante CD1d (10 ug / mL em 1x PBS) por poço e incubar durante 1 hora a 37 ° C. </li><li> Lavar quatro vezes com 200 ul de PBS 1x, adicionar 200 ul de 2% de FCS em PBS 1x (solução de bloqueamento) por poço e incubar durante 2 horas a 37 ° C. </li><li> Remover a solução de bloqueio, adicionar 200 ul de αGalCer (5 ng / ul) por poço e incubar a placa a 37 ° C durante 16 horas. [Para a preparação da solução αGalCer, adicionar 1,085 μ 1x PBS para 55 ug de αGalCer dissolvidos em veículo que contém 96 mg / mL de sacarose, 10 mg / ml de desoxicolato de sódio e 5 mg / ml de Tween 20]. </li><li> Após incubação, eliminar a solução e lavar o αGalCer bem duas vezes com 200 ul de PBS 1x seguido por 200 ul de meio completo RPMI 1640. </li><li> Recolher as células iNKT expandidas in vitro no dia 21, as células de centrifugação a 300 xg durante 10 min a 4 ° C e ressuspender em R completoPMI 1640 a uma concentração de 0,5 x 10 6 células / ml. </li><li> Adicionar 200 ul da ressuspensão iTNK célula por poço e incubar durante 72 horas a 37 ° C em uma incubadora de CO 2. </li><li> Recolhe-se o sobrenadante e medem citocinas de interesse por ensaio imunoabsorvente ligado a enzima (ELISA), de acordo com o protocolo do fabricante. </li></ol>

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B., Aspeslagh, S., Elewaut, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invariant+natural+killer+T+cells+in+rheumatic+disease:+a+joint+dilemma.">Invariant natural killer T cells in rheumatic disease: a joint dilemma.</a> Nat Rev Rheumatol. 6, 90-98 (2010).</li><li>Terabe, M., Berzofsky, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKT+cells+in+immunoregulation+of+tumor+immunity:+a+new+immunoregulatory+axis.">NKT cells in immunoregulation of tumor immunity: a new immunoregulatory axis.</a> Trends Immunol. 28, 491-496 (2007).</li><li>Molling, J. 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The authors have no competing interests to disclose.

Etapas críticas do protocolo atual incluem o isolamento e enriquecimento posterior de CD5 + linfócitos (Seção 1 &amp; 2), FACS classificação (Seção 3) eo revestimento inicial de células iNKT (Seção 4). Dos passos realizados na Seção 1, lembre-se para a camada cuidadosamente a suspensão celular splenic a médio gradiente de densidade tal que uma interfase celulares distintos é gerado após a centrifugação. O enriquecimento posterior para CD5 + linfócitos por separação magnética ...

Murino células T assassinas naturais invariante (iNKT) são uma população distinta de linfócitos T inatas selecionados no timo por expressando CD1d timócitos corticais 1,2. iNKT células expressam um receptor de células T (TCR) composta de uma cadeia Vα14-Jα18 TCR invariante emparelhado com qualquer Vβ8, Vβ7 Vβ2 TCRs ou 3, que é capaz de reconhecer endógena, bem como os antigénios de lípidos estranhos no contexto da CD1d. Por exemplo, células de murino iNKT reconhecer e são activados por um chamado lípido isoglobotrihexosylceramide antigénio endógeno (iGb3) 4, bem como α-galactosilceramida (αGalCer) 5,6, um glicolípido isolado a partir de esponjas marinhas. TCR-dependente de activação de células iNKT promove a iniciação de respostas imunitárias adaptativas, e como um resultado, as células iNKT foram mostrados para ser funcionalmente envolvidos na melhoria ou o desenvolvimento de uma variedade de patologias incluindo a doença reumática 7 e cancro &lt;sup&gt; 8. Actualmente, os ligandos celulares iTNK sintéticos constituem novos adjuvantes de vacinas promissoras, que podem ser capazes de regular a um número de condições imunopatológicas. Foi previamente demonstrado que as células iNKT podem ser gerados in vitro após isolamento a partir de tecido de rato no entanto; muitos destes estudos emprega a utilização de células primárias de apresentação de antigénio (APCs) e / ou linhas de células 9, Vα14 TCR transgénicos (Tg) de 10 ratinhos, timomas ou para a geração de iNKT derivadas de células hibridomas 11,12. Além disso, um grande número de ratos, altos volumes de reagentes, tais como dímeros CD1d αGalCer-carregados, e tempos de cultivo longas fazer alguns protocolos publicados há menos de forma ética e economicamente atraente 9,13. Neste relatório nós descrevemos um método adaptado para o isolamento e expansão in vitro de células de rato iNKT baço. Mais especificamente, o protocolo descreve um métodopara o enriquecimento de células de baço de ratinho iNKT que reduz os ratos, os reagentes e tempo necessários para FACS separação de células, e propõe uma abordagem otimizada para expansão de células esplênicas iNKT ordenados in vitro.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-2</td>
			<td>eBiosciences</td>
			<td>14-8021</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-12</td>
			<td>eBiosciences</td>
			<td>39-8122-65</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-7</td>
			<td>eBiosciences</td>
			<td>14-8071</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD3e (145-2C11)</td>
			<td>eBiosciences</td>
			<td>16-0032-86</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD28 (37.51)</td>
			<td>eBiosciences</td>
			<td>16-0281-85</td>
			<td></td>
		</tr>
		<tr>
			<td>e450-conjugated anti-mouse CD19 (eBio1D3)</td>
			<td>eBiosciences</td>
			<td>48-0193-82</td>
			<td></td>
		</tr>
		<tr>
			<td>e450-conjugated anti-mouse CD8&#945; (53-6.7)</td>
			<td>eBiosciences</td>
			<td>48-0081-82</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-conjugated anti-mouse CD5 (53-7.3)</td>
			<td>eBiosciences</td>
			<td>11-0051-81</td>
			<td></td>
		</tr>
		<tr>
			<td>V500-conjugated anti-mouse CD3&#949; (500A2)</td>
			<td>BD biosciences</td>
			<td>560771</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD5 (Ly-1) microbeads</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-049-301</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD16/32 (2.4G2)</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MS Columns</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue, 0.4% (wt/vol)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco</td>
			<td>12633-020</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Gibco</td>
			<td>10010-056</td>
			<td>[Ca2+/Mg2+ - free]</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Gibco</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-123</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4458-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>EDS-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque plus</td>
			<td>GE Healthcare</td>
			<td>71-7167-00 AF</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate F-bottom</td>
			<td>Greiner-bio one</td>
			<td>657-160</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Greiner-bio one</td>
			<td>665-180</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Greiner-bio one</td>
			<td>655-180</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml falcon tube</td>
			<td>Greiner-bio one</td>
			<td>188-271</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml falcon tube</td>
			<td>Greiner-bio one</td>
			<td>227-261</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m filter</td>
			<td>Greiner-bio one</td>
			<td>542-070</td>
			<td></td>
		</tr>
		<tr>
			<td>30 &#181;m filter</td>
			<td>Millipore</td>
			<td>SVGP01050</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS separator</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-jacketed CO2 incubator</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Kit</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSAria III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized method for isolating and expanding invariant natural killer t cells from mouse spleen

A capacidade de secretar citocinas rapidamente com a estimulação é uma característica funcional da invariante natural killer T (iNKT) linhagem celular. iNKT células são portanto caracterizado como uma população de células T capazes de activar inata e adaptativa direcção respostas imunes. O desenvolvimento de técnicas melhoradas para a cultura e expansão de células iNKT murino facilita o estudo de biologia celular iNKT in vitro e em sistemas modelo in vivo. Aqui, descrevemos um processo aperfeiçoado para o isolamento e a expansão de células esplénicas murinas iNKT. Os baços de ratinhos C57BL / 6 são removidos, dissecados e tenso e a suspensão celular resultante é colocado em camadas sobre meios de gradiente de densidade. Após centrifugação, as células mononucleares esplénicas (MNCs) são recolhidos e linfócitos CD5-CD5 positivas (+) são enriquecidos para usando esferas magnéticas. iNKT células dentro da fracção CD5 + são subsequentemente corados com ^5; GalCer-carregado CD1d tetrâmero e purificou-se por selecção de células activadas de fluorescência (FACS). Separadas por FACS, em seguida, as células são iNKT inicialmente cultivadas in vitro, utilizando uma combinação de citocinas recombinantes de murideo e receptor de células T de placa ligado (TCR), antes de ser expandido estímulos em presença de murino IL-7 recombinante. Usando esta técnica, aproximadamente 10 8 iNKT células pode ser gerada dentro de 18-20 dias de cultura, após o que eles podem ser utilizados para ensaios funcionais in vitro, ou para experiências in vivo em ratinhos de transferência.

Isolamento de células mononucleares esplénicas utilizando um gradiente de densidade leva cerca de 1 hora e elimina a utilização de reagentes necessários para a lise de glóbulos vermelhos (RBC). Um elevado rendimento de células viáveis ​​é obtida usando este método e detritos gerados durante a deformação do órgão é removido. Tipicamente, a frequência de células iNKT dentro do conjunto de linfócitos do baço varia entre 1 e 5% de linfócitos T totais no entanto, isto pode variar, dependendo do estado...

Watch this Scientific Journal Video about An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen at JoVE.com

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

Um método otimizado para o isolamento e expansão de células invariante Natural Killer T de Rato Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

an-optimized-method-for-isolating-expanding-invariant-natural-killer

Research

JoVE Journal

Immunology and Infection

11.9K visualizações.  Ghent University Hospital. Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents. 

Vídeo: An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

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 ...

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 ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Antígeno Artificial célula apresentadora de ativação mediada (AAPC) e expansão de células T Natural Killer

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Assassino Artificial células apresentadoras de antígenos (KaAPC) para a eficiente<em&gt; In Vitro</em&gt; Depleção de células T humanas antígeno específico da

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

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

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

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Citometria de fluxo e assassinas naturais celular Lytic Actividade em sangue total humano

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Um novo sistema alimentador-livre para produção em massa de murino Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Isolando os linfócitos de rato pequeno Intestinal sistema imunológico

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

Quantificação de baseados em citometria de fluxo multicolor de mitocôndrias e lisossomos em células T

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...