This work was supported by grants BFU2011-29450, BFU2008-04342/BMC from the Spanish Ministry of Science and Innovation and PIES201020I046 from Consejo Superior de Investigaciones Cient&#236;ficas (CSIC).

Nota: Os procedimentos experimentais foram aprovados pelo Comitê de Ética em Pesquisa da Universidad Autónoma de Madrid e conduzido sob a supervisão da Universidad Autónoma de Madrid Cabeça de Bem-Estar Animal e Saúde de acordo com diretrizes espanholas e europeias. Os camundongos foram criados em free (SPF) de habitação específico patógeno e eles foram sacrificados por pessoal treinado e qualificado, utilizando dióxido de carbono (CO 2) método de inalação. 1. Rato Medula Óssea derivado DCs Differentiation and Infection Nota: A Figura 1 resume este primeiro passo. Todos os procedimentos devem ser realizados na capa deste ponto em diante, usando apenas estéreis mídia, instrumentos, pontas para pipetas e placas de cultura. <ol><li> Rato de Medula Óssea DCs derivadas de Diferenciação <ol><li> Dissecar tíbias e fémures de uma C57 / BL6 rato 22 e transferir para um prato de plástico com 10 ml de meio RPMI. </li&gt; <li> Corte cada epífise off, com tesoura estéril e expor medula óssea, que tem uma cor brilhante vermelha característica. </li><li> Infundir o interior do osso com 10 ml de solução salina tamponada com fosfato (PBS) contendo 0,5% de albumina de soro bovino (BSA) e 5 mM de ácido etilenodiaminotetracético (EDTA) (tampão / BSA / EDTA PBS) utilizando uma seringa esterilizada em um Petri estéril prato. </li><li> Recolha a suspensão celular e centrifugue num tubo a 600 xg numa centrifugadora refrigerada (a 4 ° C). </li><li> Ressuspender as células em 1 ml de cloreto de amónio-potássio-(ACK) tampão de lise (0,15 M NH 4 Cl, 10 mM KHCO 3 e EDTA 0,1 mM) e incuba-se durante 30 segundos a temperatura ambiente, a fim de eliminar as células vermelhas do sangue. </li><li> Adicionar 10 ml de meio RPMI 1640 com 10% de soro fetal de bovino (FBS) e filtrar através de um filtro de células (70 ^ M) para remover os fragmentos ósseos e aglomerados de células antes da centrifugação com a mesma velocidade (600 xg) porque aglomerados foram eliminados por filtração. </li><li> Contar as células umad ajustar para 5 x 10 5 células / ml de RPMI com 10% de FBS (contendo 50 ^ M B-mercaptoetanol, piruvato de sódio 1 mM) e adiciona-se 20 ng / ml de factor recombinante de murino de granulócitos-macrófagos de colônias estimulante (RM-GM-CSF) como uma concentração final. </li><li> Adicionar a esta suspensão, uma qualidade microbiológica estéril, 15 cm numa caixa de Petri, e a cultura num incubador de CO2 (37 ° C, 5% CO 2). </li><li> De três em três dias, girar ambas as células não aderentes e as células destacadas com EDTA 5 mM em PBS e ressuspender em 5 x 10 5 culas / ml de densidade celular, com meio fresco contendo 20 ng / ml de RM-GM-CSF. </li></ol></li><li> Maturação das DCs e Antigen Carregando <ol><li> No dia 9, ressuspender as células a 5 x 10 5 culas / ml de densidade em meio contendo 20 ng / ml de RM-GM-CSF. Em seguida, adiciona-se 20 ng / ml de lipopolissacárido (LPS) para permitir a expressão elevada de MHC-II em células e incuba-se um não-tecido tratadas com cultura estéril prato de Petri 15 cm durante 24 horas. </li><li> Depois de umdia de LPS-estimulação, manchar algumas células com anticorpos contra DC CD11c rato, MHCII e Gr-1 para buscar diferenciação DCs por citometria de fluxo. Um exemplo de análise de citometria de fluxo de DC é mostrado na Figura 3A e 3B. <ol><li> Incubar as DCs (5 x 10 5 células / amostra) com um anticorpo monoclonal anti-ratinho CD16 / CD32 a 2,5 ug / ml de concentração final em 50 ul de PBS / BSA / EDTA por amostra. Em seguida, adicionar 50 ul de anticorpos contra os MHCII (IA / IE), CD11c e Gr-1 conjugados com diferentes fluorocromos para 1: 100, 1: 200 e 1: 500 em / tampão de BSA / PBS EDTA respectivamente. </li><li> Finalmente, lavar com DCs / tampão de BSA / PBS EDTA e analisar por citometria de fluxo de acordo com o protocolo do fabricante. Nota: Se as células são bem diferenciados, células estão prontas para carga de antigénio. </li></ol></li><li> Lavar as DCs com meio RPMI 10% FBS (sem antibióticos) e incubar-los com 10 ug / ml OTII OVAp (OVA 323-339; ISQAVHAAHAEINEAGR) em um tubo de plástico em 1 ml de RPMI 10% FBS por 5 x 10 6 DC mínimo para 1 h a 37 ºC. Como controlo negativo, deixe DCs sem incubação com OVAp. </li></ol></li><li> A infecção de DCs <ol><li> Infect DCs a uma multiplicidade de infecção (MOI) de 10 bactérias (10 bactérias por DC) durante 1 hora numa incubadora de CO2 (37 ° C, 5% CO 2). </li><li> Lavar as células DC 3x em PBS 1x e em RPMI 10% FBS e centrifugar a 450 xg de modo a lavar a maior parte das bactérias extracelulares. Finalmente, Ressuspender as células em RPMI 10% FBS (sem antibióticos) a 20 x 10 6 células / ml. </li></ol></li></ol> 2. Isolamento de linfócitos T CD4 + a partir de Ratos Transgénicos OTII Nota: A Figura 1 resume esta segunda etapa. Os gânglios linfáticos deve ser usado em vez de baço para isolar as células T CD4 +, pois a proporção de linfócitos T CD4 + no nódulo linfáticos (~ 50%) é maior do que no baço (~ 25%) e, portanto, a purificação poderia ser mais eficaz. <ol><li> Faça suspensão de células individuais de linfonodos <ol><li> Remover inguinal, axilar, braquial, do colo do útero e da linfa mesentéricos 23 a partir de ratinhos transgénicos OTII e transferir para um prato de plástico com 10 ml de RPMI 10% de FBS. </li><li> Linfonodos moagem sob um capuz estéril usando duas lâminas de microscópio fosco. Coloque gânglios linfáticos de um lado fosco de uma lâmina de microscópio, e esfregue com o lado fosco do segundo slide até órgãos têm sido chão. </li><li> Lavar única suspensão celular em tampão PBS / BSA / EDTA. </li><li> Filtrar as células embora um filtro de células (70 ^ M) para remover o tecido conjuntivo e lava-se com / tampão de BSA / PBS EDTA. </li><li> Ressuspender as células em 1 ml de tampão de lise ACK e incubar durante 1 min à temperatura ambiente a fim de eliminar as células vermelhas do sangue. </li></ol></li><li> Isolamento de células T CD4 + <ol><li> Lave novamente como no passo 2.1.3 e contagemcélulas para voltar a suspender-los em 100 x 10 6 células / ml em / tampão de BSA / PBS EDTA. Adicionar anticorpos biotinilados contra CD8, IgM, B220, CD19, MHC de classe II (I-Ab), CD11b, CD11c e DX5 a 1: 250 durante 30 min em gelo para posterior selecção negativa de células T CD4 +. </li><li> Lave as células em / BSA / EDTA PBS e incubar as células (100 x 10 6 células / ml) com microesferas de estreptavidina na concentração recomendada para o protocolo do fabricante, durante 15 min em gelo. </li><li> Lave as células em / tampão de BSA / PBS EDTA e filtrá-las, embora de um filtro celular (30 uM) antes do isolamento de células T CD4 +. </li><li> Isolar as células T CD4 + por selecção negativa utilizando uma máquina de separação de células magnético de acordo com o protocolo do fabricante. </li><li> Contar as células e ajusta-se 4 x 10 6 culas / ml com meio RPMI 10% FBS (sem antibióticos). </li><li> Corrigir e manter em gelo isolaram células T CD4 + (3 x 10 5 células) para verificar a pureza por fluxocitómetro como descrito 24. Um exemplo de células T CD4 + de purificação é mostrada na Figura 3C. </li></ol></li></ol> 3. T celular Transinfection Medição por ensaio de proteção gentamicina Nota: A Figura 2 resume esta terceira etapa do protocolo. <ol><li> T Transinfection celular <ol><li> Incubar as células T CD4 + isoladas com células infectadas DC (1: 1) (as DCs foram infectadas durante 1 hora e lavou-se para eliminar as bactérias livres) durante 30 min para permitir a formação de sinapses imune em um incubador de CO2 (37 ° C, 5% CO 2). </li><li> Adicionar 0,5 ml de células T CD4 + isoladas (2 x 10 6 células) e 0,1 ml de DCs infectadas (2 x 10 6 células) numa placa de cultura de 24 poços. Como controlo negativo, infectar directamente 0,5 ml de células T CD4 + a uma MOI de 10 bactérias durante 30 min. </li><li> Incluir controlos adicionais que separam DCs infectadas a partir de células T utilizandouma barreira de policarbonato Transwell (com 3 um de tamanho dos poros), impedindo o contacto físico DC-T. Adicionar 0,1 ml de DC dentro do transwell e 0,5 ml de células T CD4 + para dentro do compartimento inferior do poço (placa de 24 poços). </li><li> Finalmente, com meio RPMI completo até que não é de 0,6 ml em cada poço para tornar volumes iguais em todas as amostras. </li><li> Após 30 minutos de formação da sinapse imune, adicionar 100 ug / ml de gentamicina e incubar durante 1 h antes de se recolher as células numa incubadora de CO2 (37 ° C, 5% CO 2). </li></ol></li><li> Re-isolamento de células T CD4 + <ol><li> Recolher as células não aderentes e ressuspender-los em tampão de PBS / BSA / EDTA. A maioria das DCs permanecem ligadas à placa de cultura de plástico. Tubos de vórtice suavemente para garantir a desagregação celular. Manter a 500 ul de sobrenadante de células como controlo para mostrar que a gentamicina tem funcionado bem. </li><li> Re-isolamento das células T CD4 + a partir de amostras que contêm células T e DCs em conjunto, como no passo 2.2, Embora a maioria dos DCs deveria ter anexado ao prato. Mantenha as amostras de controlo no gelo. Leve em conta apenas experimentos com menos de 2% de contaminação. Um exemplo de células T CD4 +, após purificação transinfection é mostrado na Figura 3D. </li></ol></li><li> As células T Lyse e semeá-los em bactérias placas de meio-ágar. <ol><li> Lave as células T CD4 + por duas vezes com PBS. </li><li> Contar as células e ressuspender-los a 2 x 10 6 culas / ml em PBS. </li><li> Adicionar 500 ul de 0,1% de Triton X-100 em 500 uL de células T para lisar-los a 1 x 10 6 células / ml, liberando as bactérias intracelulares para fora das células T. </li><li> Faça 3 diluições decimais seriadas de células lisadas e sementes 50 ul de cada diluição em placas de ágar bactérias médios. Divida placas em 4 porções e semear as diferentes diluições de células lisadas em cada parte. Os resultados representativos são mostrados na Figura 4. <ol><li> Como controlo, também a placasobrenadante de células armazenados dos conjugados em agar, para assegurar que a gentamicina tem funcionado bem. Plate, também DCs infectadas como um controle adicional. No caso de baixa contaminação ocorre DCs (&lt;2%), subtrair as unidades formadoras de colónias (CFUs) correspondentes a baixa contaminação DC. </li></ol></li></ol></li></ol> 4. Quantificação de células T Transinfection por citometria de fluxo Nota: A Figura 2 sumaria este passo. <ol><li> T Transinfection celular <ol><li> Infect BMDCs como na etapa 1.3, mas usar bactérias expressando GFP neste caso. </li><li> Mancha células T CD4 + (isoladas de nódulos linfáticos no passo 2.2) com um rastreador aminocoumarin célula clorometilo (CMAC) durante 30 min a 37 ºC de acordo com o protocolo do fabricante. </li><li> Lave as células T CD4 + rotulados duas vezes com RPMI a 10% de FBS (sem antibióticos). </li><li> Incubar as DCs infectadas (após 1 h de incubação com bactérias) com rotulados CD4 +As células T durante 30 minutos para permitir a formação de sinapses imunes a 37 ° C, incluindo todos os controlos que explicam no passo 3.1.1. </li></ol></li><li> Coloração de células infectadas T CD4 + <ol><li> Após 30 min de DC-T a formação de sinapses imune, recolher as células e lava-las em PBS / BSA / EDTA. </li><li> Após duas lavagens com tampão PBS / BSA / EDTA, células agregados separados por vórtice suavemente os tubos. </li><li> Fixar as amostras de 0,2 ml / tubo de PBS contendo 3% de PFA durante 15 min à TA. Em seguida, lave-os em PBS. </li><li> Incubar com anticorpo monoclonal anti-ratinho-CD16 / CD32 (2,5 ug / ml) em 50 ul / amostra de PBS / BSA / EDTA durante 15 min em gelo para bloquear os receptores Fc de células dendríticas. </li><li> Adicionar um coelho anti-bactérias anticorpo (10 ug / ml como concentração final) em 50 ul / amostra de PBS / BSA / EDTA durante 30 min em gelo para corar bactérias extracelulares. </li><li> Lavar as células com PBS / BSA / EDTA e incubar com um anticorpo contra CD11c rato conjugado com ficoeritrina (PE) e uma SEcundária anticorpo contra coelho conjugado com outro fluorocromo Alexa-Fluor como 647 a 1: 200 em 100 ul / amostra de PBS / BSA / EDTA durante 30 min em gelo. </li><li> Lave as células e analisar as amostras por citometria de fluxo. Não se esqueça de incluir uma amostra de células T transinfected usando bactérias não fluorescentes como controle negativo e amostras marcadas com apenas um de cada fluorocromo utilizado no experimento para compensar as amostras e ajustar a citometria de fluxo configurações 25. Análise representativa está mostrada na Figura 5. Nota: Se as bactérias não expressam GFP, para distinguir intracelular e bactérias extracelulares, bactérias extracelulares etiqueta, corrigir, e permeabilizar amostras com Triton X-100 a 0,5% em PBS durante 5 min. Depois corar bactérias intracelulares totais (+ extracelular) com um fluorocromo diferente. </li></ol></li></ol>

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The authors have nothing to disclose.

As células T ou linfócitos T são um tipo de leucócitos que desempenham um papel central na imunidade mediada por células e pertencem a resposta imune adaptativa 26. As células T são refractários à infecção in vitro, mas alguns relatórios indicam que eles podem ser infectadas in vivo 14,15. Os contatos íntimos de células T e APC durante sinapse imune servir de plataforma para o intercâmbio de material biológico 13, incluindo alguns vírus, como o HIV <sup...

Quando um agente patogénico infecta o seu hospedeiro, geralmente há uma ativação da resposta imune inata e adaptativa, necessários para o apuramento bacteriana. A imunidade inata é a primeira linha de defesa que impede a maioria das infecções. A imunidade inata em distinguir a maneira como os elementos precisos que são conservadas entre grandes grupos de microrganismos (padrões moleculares associados a agentes patogénicos, PAMPS 1). Os mecanismos da imunidade inata incluem barreiras físicas tais como a pele, as barreiras químicas (péptidos antimicrobianos, lisozima) e os leucócitos inatos, que incluem os fagócitos (macrófagos, neutrófilos, e células dendríticas), mastócitos, eosinófilos, basófilos e células assassinas naturais 2. Estas células identificar e eliminar patógenos, quer por atacá-los através do contato ou via fagocitose, que inclui patógeno engolindo e matando. Este sistema não permite a defesa ao longo da vida, em contraste com a imunidade adaptativa, que conferem memória imunológica contra pathogens. O sistema imune adaptativo é a segunda linha de defesa e é capaz de reconhecer e de reagir a antigénios específicos de múltiplos microbiana e substâncias não-microbianos 3. Os principais componentes do sistema imunitário adaptativo são linfócitos, que incluem células B e T. As células B estão envolvidos na resposta humoral, que segregam anticorpos contra os agentes patogénicos ou proteínas exógenos. No entanto, as células T representar a imunidade mediada por células, modulando a resposta imunitária com citocinas secreção ou matando as células infectadas com agentes patogénicos 4. Células apresentadoras de antígenos (APCs) incluindo os macrófagos ou células dendríticas, dos componentes do sistema imune inato, pode reconhecer patogénios fagocitam e processos componentes bacterianos em antigénios, que são apresentados na superfície da célula pelo complexo principal de histocompatibilidade (MHC) de 5-7. Depois de APCs foram fagocitados patogénios, eles usualmente migram para os gânglios linfáticos de drenagem, onde eles interagem com Tcélulas. Os linfócitos T podem reconhecer complexos MHC-péptido específicos pelos seus receptores de células T. A sinapse imune (IS) ocorre na interface entre uma APC carregadas com antigénio e um linfócito durante a apresentação do antigénio 8,9. Algumas bactérias podem sobreviver fagocitose e divulgar sistematicamente dentro APCs. Nesta visão, APCs infectados servem como reservatórios de bactérias ou &quot;cavalos de tróia&quot; que facilitam a propagação de bactérias 10. O contato íntimo entre APCs e os linfócitos que ocorrem durante o curso de formação é também funcionar como uma plataforma para a troca de parte das membranas, material genético e exossomos e pode ser sequestrado por alguns vírus para infectar células T; este processo é chamado transinfection 11-13. Algumas bactérias patogénicas (Listeria monocytogenes, Salmonella enterica e Shigella flexneri) são capazes de invadir os linfócitos T in vivo e alterar o seu comportamento 14-16. Nós temosdescrito recentemente que os linfócitos T também são capazes de capturar as bactérias por transinfection a partir de células dendríticas infectadas anteriormente (DCs) durante o curso de apresentação de antigénio 16. T célula bacteriana captura por transinfection muito mais eficaz (1,000-4,000x) do que infecções diretos. T patógeno células captura e bactérias não-patógeno indicando que transinfection é um processo impulsionado por células T. Surpreendentemente, transinfected T (TIT) células mortas rapidamente as bactérias capturados e fê-lo de forma mais eficiente do que os fagócitos profissionais 16. Estes resultados, que quebram um dogma de imunologia, mostram que as células de imunidade adaptativa pode executar funções que foram supostamente exclusiva da imunidade inata. Além disso, mostrámos que as células tit secretar grandes quantidades de citocinas pró-inflamatórias e proteger de infecções bacterianas in vivo. Aqui apresentamos os diferentes protocolos utilizados para estudar o processo transinfection bacteriana emum modelo de rato. Este modelo baseia-se na utilização de células T CD4 + a partir de ratinhos transgénicos OTII, que carregam um TCR específico para o péptido 323-339 de OVA (OVAp) no contexto de I-Ab 17 que interagem especificamente com o osso bacteriana infectada marrow- DCs derivadas BMDCs (18,19) carregados com OVAp, que formam sinapses imunes estáveis. T transinfection célula pode ser visualizado e rastreados utilizando microscopia de fluorescência. Além disso, a citometria de fluxo pode ser usado para a detecção de células infectadas por aproveitando a fluorescência emitida por bactérias que expressam a proteína fluorescente verde (GFP) 16,20. Além disso, T transinfection célula pode ser quantificada por um método mais sensível, a gentamicina ensaio de sobrevivência que permite a medição de um grande número de eventos. A gentamicina é um antibiótico que não podem penetrar nas células eucarióticas. Portanto, utilizando este antibiótico permite a diferenciação de bactérias intracelulares que sobreviveram à adição de antibiótico FRos extracelulares om que foram mortas 21.

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">RPMI</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:104px;">SH3025501</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">r-GMCSF</td>
			<td style="width:131px;">Peprotech</td>
			<td style="width:104px;">315-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">LPS</td>
			<td style="width:131px;">SIGMA</td>
			<td style="width:104px;">L2630-10mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Na Pyruvate</td>
			<td style="width:131px;">Thermo Scientific</td>
			<td style="width:104px;">SH3023901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">2-ME</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:104px;">31350-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">OVAp OTII (323&#8211;339)</td>
			<td style="width:131px;">GenScript</td>
			<td style="width:104px;">---</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Cell Strainer 70uM</td>
			<td style="width:131px;">BD</td>
			<td align="right" style="width:104px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;30uM Syringe Filcons Sterile</td>
			<td>BD</td>
			<td align="right">340598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">AutoMacs Classic</td>
			<td style="width:131px;">Miltenyi Biotec</td>
			<td style="width:104px;">130-088-887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Gentamicin</td>
			<td style="width:131px;">Normon</td>
			<td style="width:104px;">624601.6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Transwell</td>
			<td style="width:131px;">Costar</td>
			<td align="right" style="width:104px;">3415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">LB</td>
			<td style="width:131px;">Pronadisa</td>
			<td align="right">1231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Agar</td>
			<td style="width:131px;">Pronadisa</td>
			<td align="right">1800</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:341px;">Paraformaldehyde 16%</td>
			<td style="width:131px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:104px;">15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Triton X-100</td>
			<td style="width:131px;"></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD8 biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">IgM Biot</td>
			<td style="width:131px;">ImmunoStep</td>
			<td style="width:104px;">Clone RMM-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">B220 Biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD19 biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right">553784</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">MHC-II Biot (I-A/I-E)</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right">553622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD11b biot</td>
			<td style="width:131px;">Immunostep</td>
			<td style="width:104px;">11BB-01mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD11c biot</td>
			<td style="width:131px;">Immunostep</td>
			<td>11CB3-01mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">DX5 biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Gr-1 biot</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD16/CD32</td>
			<td style="width:131px;">ImmunoStep</td>
			<td>M16PU-05MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">anti Salmonella</td>
			<td style="width:131px;">ABD Serotec</td>
			<td style="width:104px;">8209-4006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD11cPE</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right" style="width:104px;">553802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CD4-APC</td>
			<td style="width:131px;">Tonbo Biosciences</td>
			<td style="width:104px;">20-0041-U100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gr-1 APC</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right">553129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MHC-II (I-A/I-E) FITC</td>
			<td style="width:131px;">BD Biosciences</td>
			<td align="right">553623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa-Fluor 647 Goat Anti-Rabbit IgG (H+L) Antibody, highly cross-adsorbed</td>
			<td>Invitrogen</td>
			<td>A-21245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">CMAC (7-amino-4-chloromethylcoumarin)</td>
			<td style="width:131px;">Life technologies</td>
			<td>C2110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">BSA</td>
			<td style="width:131px;">SIGMA</td>
			<td style="width:104px;">A7030-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Streptavidin MicroBeads</td>
			<td style="width:131px;">Miltenyi Biotec</td>
			<td style="width:104px;">130-048-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">BD FACSCanto II</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:104px;">---</td>
			<td></td>
		</tr>
	</tbody>
</table>

t cells capture bacteria by transinfection from dendritic cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Aqui descrevemos como executar transinfection bacteriana de células T de murino a partir de medula óssea derivadas de DCs infectadas e como medir transinfection bacteriana através de duas abordagens diferentes: a citometria de fluxo e ensaio de sobrevivência gentamicina A Figura 1 resume o procedimento para obter as células.. DCs são geradas por incubação de células de medula óssea com GM-CSF durante 9 dias. Em seguida, as DCs são maturados com LPS para aument...

Watch this Scientific Journal Video about T Cells Capture Bacteria by Transinfection from Dendritic Cells at JoVE.com

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Aqui é apresentado um protocolo para medir a captura de bactérias por células T CD4 +, o qual ocorre durante a apresentação de antigénio através de células dendríticas transinfection pré-infectadas (DC). Mostramos como executar os passos necessários: isolamento de células primárias, infecção de formação conjugado celular DC, DC / T, e medição da transfecção de células T bacteriana.

As células T Capturar Bactérias por Transinfection das dendríticas

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

t-cells-capture-bacteria-by-transinfection-from-dendritic-cells

Research

JoVE Journal

Immunology and Infection

11.8K visualizações.  Spanish National Biotechnology Centre (CNB-CSIC).  Aqui é apresentado um protocolo para medir a captura de bactérias por células T CD4 +, o qual ocorre durante a apresentação de antigénio através de células dendríticas transinfection pré-infectadas (DC). Mostramos como executar os passos necessários: isolamento de células primárias, infecção de formação conjugado celular DC, DC / T, e medição da transfecção de células T bacteriana. 

Vídeo: T Cells Capture Bacteria by Transinfection from Dendritic Cells

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

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

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

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

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

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

Imunologia e Infecção

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Isolamento de células dendríticas pulmonares do rato

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

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

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

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Isolamento e caracterização de células dendríticas e macrófagos a partir do intestino do rato

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Isolar e imunocoloração linfócitos e células dendríticas a partir de placas de Peyer Murino

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

O isolamento de células dendríticas mielóides e células epiteliais de timo humano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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.

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 ...

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 ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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

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

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

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

As células T Capturar Bactérias por Transinfection das dendríticas

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