Este trabalho foi financiado pela Associação Americana de Gastroenterologia (AGA) Research Award Scholars e Doença de Crohn e Colite Foundation of America (CCFA) Desenvolvimento de Carreira Award (a ENS), NIH NIDDK F30 DK089692 (a ECS) e University of North Carolina Centro de Biologia Gastrointestinal e Doença Grant P30 DK34987 (Histologia Core). O Mecanismo UNC Citometria de Fluxo Core é apoiado em parte por uma NCI Centro de Suporte Grant Núcleo (P30CA016086) à Comprehensive Cancer Center UNC Lineberger. Agradecemos Luke B. Borst da North Carolina State University College de Medicina Veterinária por sua ajuda com a análise histopatológica e imuno-histoquímica.

NOTA: Certifique-se que todos os protocolos de animais são aprovados por e em conformidade com Institutional Animal Care e do Comitê Use regulamentos (IACUC) e no Guia do Conselho Nacional de Pesquisa para a Conservação e Uso de Animais de Laboratório. Camundongos doadores podem ter sexo masculino ou feminino, mas ratos destinatário devem ser do sexo masculino. Se os destinatários do sexo feminino devem ser utilizados, os camundongos doadores têm que ser fêmeas 5. Manter colónias usando, roupa de cama não estéril normal e água não acidificado, uma vez que estes podem causar impacto na microbiota intestinal, e, portanto, o fenótipo dos ratinhos colite 5,6. 1. Preparação Experimental <ol><li> Use mídia e buffers geladas. Mantenha as células no gelo durante todo o experimento. </li><li> Realizar o experimento na capa de risco biológico estéril usando técnica estéril. </li></ol> 2. Isolamento de células T do baço <ol><li> Sacrificá camundongo doador / mice em CO 2 câmara seguido por dislocatio cervicaln. Spray de abdômen com etanol 70%. </li><li> Faça uma incisão horizontal no abdômen e descascar a pele para expor peritônio. Segure peritônio longe dos órgãos internos com a pinça e fazer uma incisão no peritônio abdominal esquerda para expor e extirpar o baço. </li><li> Inserir o baço em 10 ml de Meios completa em uma placa de Petri. Retire e elimine o excesso de tecido do baço. </li><li> Use duas lâminas de vidro esterilizados para esmagar e provocar uma separação baço em suspensão de uma única célula. Suspensão de células Filtrar através de um filtro de 70 mm em um tubo de 50 ml e lavar filtro com 5 ml de Meio Completo. Coloque até 5 baços em um tubo de 50 ml cônico. </li><li> Centrifugar células a 450 xg por 7 min. Elimine o sobrenadante por vazamento off ou por sucção a vácuo em um recipiente de resíduos. </li><li> Suavemente ressuspender as células em 5 ml por baço de um tampão de lise, para lisar as células vermelhas do sangue durante 10 min à temperatura ambiente. Adicionar um volume igual de meio completo (5 ml por baço) para o tubo. </li><li&gt; células centrifugar a 450 xg por 7 min. Elimine o sobrenadante por vazamento off ou por sucção a vácuo em um recipiente de resíduos. </li><li> Volte a suspender suavemente as células em 10 ml de Etiquetagem Buffer. </li><li> Contagem de células por exclusão de azul de tripano. <ol><li> Retirar 20 l de suspensão de células e adicionar 180 mL de azul tripano para um tubo de microcentrífuga e misture bem. Após 5 min, adicionar 10 ul de células e contagem em hemocitómetro células não-azul sob o microscópio rotulados. Determinar o número total de células viáveis. Descarte de células / trypan azul mix. </li></ol></li><li> Centrifugar células em 10 ml de Etiquetagem tampão a 450 xg por 7 min. Elimine o sobrenadante por vazamento off ou por sucção a vácuo em um recipiente de resíduos. </li></ol> 3. O enriquecimento de células T CD4 + NOTA: Siga as instruções do fabricante para produtos específicos utilizados nesta seção. <ol><li> Suavemente ressuspender as células a uma suspensão de uma única célula de 20 x 10 6 células / ml em Etiqueta frioing Buffer. </li><li> Adicionar 5 mL por 1 x 10 6 células de anticorpos de enriquecimento de células T CD4 bioestanhados. Incubar as células em gelo durante 15 min. </li><li> Adicionar 10x volume de Etiquetagem Buffer. Centrifugar células a 450 × g por 7 min. Aspirar cuidadosamente todo o sobrenadante usando sucção a vácuo em um recipiente de resíduos. </li><li> Absolutamente vortex partículas magnéticas de estreptavidina conjugada. Adiciona-se 5 ul de partículas por 1 x 10 6 células. </li><li> Misture bem, mas com cuidado. Mantenha a mistura a 6-12 ° C durante 30 min. </li><li> Adicionar Etiquetagem Tampão a uma concentração de 20-80 x 10 6 células / ml. Transferir-se para 1,0 ml de células marcadas por 12 x 75 mm Tubo de ensaio de fundo redondo (referido como o &quot;tubo-fracção positiva&quot;). </li><li> Coloque cada tubo fração positivo no ímã para 6-8 min. </li><li> Com os tubos de fração positiva ainda no ímã, transferir cuidadosamente sobrenadante do tubo-fração positiva com Pasteur de vidro pipeta para uma nova estéreis tubo de 50 ml (referred como a &quot;fracção enriquecida&quot;). Esta fração enriquecida contém células T CD4 +. Tenha cuidado para não perturbar as células marcadas atraídos pelo ímã. </li><li> Suspenda as células deixaram nos tubos-fração positiva no mesmo volume de Etiquetagem tampão como no passo 3,6 por pipetagem cima e para baixo vigorosamente. Coloque tube-fração positiva para trás em ímã para 6-8 min. </li><li> Com tubos de fração positiva ainda no ímã, transferir cuidadosamente sobrenadante (fração enriquecida, CD4 +) a partir de tubo-fração positiva com vidro Pasteur pipeta para estéril tubo de 50 ml a partir do passo 3.8, sem perturbar as células unidas a ímã. </li><li> Repita os passos 3,9-3,10 para aumentar o rendimento de células CD4 + T obtidas. Continuar protocolo utilizando fracção enriquecida (células CD4 +). </li></ol> 4. Rotulagem e Classificando Cells 7 <ol><li> Centrífuga enriquecido células a 450 xg por 7 min. Elimine o sobrenadante por vazamento off ou suct vácuoíon no recipiente de resíduos. </li><li> Ressuspender as células em 1 ml de Etiquetagem buffer. Remover alíquota de células para contar, para avaliar a viabilidade celular por exclusão com azul de tripano como no passo 2.9. </li><li> Adicionar um volume de Etiquetagem Buffer para 10 x 10 6 células / ml; se as células já estão &lt;10 x 10 6, centrifugar a 450 g durante 7 min, elimine o sobrenadante por vazamento off ou de sucção a vácuo em um recipiente de resíduos, e adicionar volume de Etiquetagem Buffer para 10 x 10 6 células / ml. <ol><li> Configure alíquotas separadas de aproximadamente 5-10 × 10 5 células cada para células de controlo não coradas, manchada de isótipo, e single-coradas em tubos de microcentrífuga. </li></ol></li><li> Adicionam-se 5 ug / ml de CD4-FITC e 1 ug / ml a CD45RB-PE para as células. Adicionar manchas de controlo de isotipo e as manchas individuais com a mesma concentração de alíquotas em tubos de microcentrífuga apropriados. Misture bem, mas com cuidado e incubar em gelo protegida da luz durante 30 min. </li><li> Adicionar 10x volume de Etiquetagem Buffer para células e controles. Centrífuga 450 g durante 7 min. Elimine o sobrenadante por sucção a vácuo em um recipiente de resíduos. </li><li> Ressuspender em Labeling Buffer para o volume na etapa 4.5. Centrífuga 450 g durante 7 min. Elimine o sobrenadante por sucção a vácuo em um recipiente de resíduos. </li><li> Ressuspender em Labeling Buffer para 10 x 10 6 células / ml. Passa através de células 70 um filtro em tubo FACS. Mantenha no gelo protegido da luz até que esteja pronto para FACS. </li><li> Configure e determinar a compensação apropriada no separador de células com células não coradas e controles individuais manchada. </li><li> Excluir células não viáveis ​​com forwarder e side-dispersão gating (Figura 1A). Configure gating para células CD45RB + CD4 + e com controles manchada de isótipo. Células portão à população CD4 +. </li><li> Ordenar CD4 + células em CD45RB alta e CD45RB populações de baixa usando um histograma simples para as células marcadas com PE em tubos com 2 ml de Meio Completo. O CD45RB alta </sup&gt; população representa o mais alto de 40% das células T CD4 + CD45RB + células (CD45RB alta), e a população de baixa CD45RB é o mais baixo de 20% das células CD4 + CD45RB + (CD45RB baixo; Figura 1B). </li></ol><img alt="Figura 1" src="/files/ftp_upload/52533/52533fig1.jpg" /> Figura 1: citometria de fluxo representativas parcelas de populações de células T CD4 + CD45RB durante a análise FACS (A - C) com FITC e CD4-PE esplenócitos CD45RB-corados de dadores murganhos C57BL / 6 foram classificadas por FACS em células CD4 + CD45RB high e CD4 +. populações de células T CD45RB baixo. Eventos (A) Doublet foram excluídos do gráfico de dispersão para a frente. (B) Os linfócitos foram fechado na trama dispersão frontal e lateral. (C) CD4 + </sup&gt; células T foram fechado, e células (D) CD4 + CD45RB + T foram plotados em um PE contra Eventos histograma. As células T CD4 + CD45RB baixas foram consideradas ser a mais baixa de 20% de células + CD45RB. As células T CD4 + CD45RB elevados foram definidos como a mais alta de 40% de células + CD45RB. <ol start="11"><li> Executar uma alíquota de cada população de células sobre a máquina de FACS para avaliar a pureza das populações. </li><li> Centrífuga classificadas células a 450 xg por 7 min. Ressuspender em 1 ml de PBS. Remover alíquota de células para contar, para avaliar a viabilidade celular por exclusão com azul de tripano como no passo 2.9. </li></ol> 5. injeção de células em Destinatários <ol><li> Ressuspender classificados células a 4 x 10 6 células / ml (alta CD45RB) ou 2 x 10 6 células / ml (CD45RB baixo) em PBS. </li><li> Transferir 100 ml de células de alta CD45RB por beneficiário para novo tubo estéril. Adicionar 100 ul de PBS pordestinatário a este tubo. Assim, o volume total de injecção por animal é de 200 l, e valor total de células por beneficiário é de 4 x 10 5 CD4 + CD45RB células T naive elevados. </li><li> Se o grupo experimental receber células T reguladoras é desejada, transferir 100 ml de células de alta CD45RB por beneficiário para novo tubo estéril. Adicionar 100 ul de células de baixa CD45RB por recipiente para o mesmo tubo. O volume total de injecção por animal é de 200 uL; proporção de CD45RB alta: células de baixa CD45RB é 2: 1. </li><li> Injectar 100 ul de CD45RB alta ou CD45RB alta CD45RB baixo CD4 + células / intraperitoneal em o lado direito e esquerdo do abdômen (total de 200 ul) de cada destinatário. </li></ol> 6. Monitoramento da progressão da doença em animais receptores <ol><li> Para avaliar o estado clínico dos animais receptores, atribuir escores clínicos agregados para o seguinte parameters 8 no dia da injecção, decorrida uma semana, e no momento do sacrifício: <ol><li> Determine desperdiçando medindo a perda de peso: 0 - sem perda de peso; Perda de 0,1-10% do peso corporal inicial - 1; 2 - A perda de mais de 10% do peso corporal inicial (Figura 2A). </li></ol></li></ol><img alt="Figura 2" src="/files/ftp_upload/52533/52533fig2highres.jpg" width="650" /> Figura 2: sinais patológicos clínicos e brutas de inflamação ocorrem após a transferência de tipo selvagem CD4 + CD45RB células T em alta RAG1 - / - e ratos destinatário NRDKO 11 (a) destinatários NRDKO perderam, em média, 10% do seu peso corporal inicial por 5. semanas após a transferência, enquanto RAG1- / - Os beneficiários não apresentaram sinais clínicos da doença neste momento. Cada ponto representa a média percentual do peso corporal inicial para a coorte ± SEM. **, P &lt;0,005. (B) Alguns ratos desenvolveram inflamação intestinal grave, como demonstrado pela presença de prolapso retal. Esta é uma imagem representativa de prolapso retal em um mouse destinatário NRDKO. (C) Grosseiramente, a partir de dois pontos, tanto RAG1 - / - e ratinhos receptores NRDKO são espessadas e menor do que os dois pontos de RAG1 - / - e ratinhos NRDKO sem transferência adoptiva de células T. Colons de ratos receptores NRDKO mostrar inflamação grave e aumentou o peso dos dois pontos (dados não mostrados). <a href="https://www.jove.com/files/ftp_upload/52533/52533fig2highres.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a> <ol><li><ol start="2"><li> Determinar a qualidade das fezes dos animais, colocando em recipiente limpo até que ele passoubanquinho: 0 - none; 1 - fezes moles; 2 - fezes aquosas e / ou com sangue. </li><li> Determinar a saúde geral do animal através presença dos seguintes sinais da doença: 0 - nenhuma postura arqueada, eriçou pele, ou pele lesões; 1 - qualquer um de presente o seguinte: debruçado postura, eriçou pele, ou lesões de pele. </li><li> Determinar a presença de prolapso retal: 0 - ausente; 1 - presente (Figura 2B). </li></ol></li><li> Sacrificar animais por inalação de CO2 seguido por deslocamento cervical, quando eles perderam 20% do seu peso corporal inicial ou no ponto de tempo desejado, o que ocorrer primeiro. A doença clínica é tipicamente evidente a partir de 5 semanas de pós-repletion. </li><li> Avaliar para a gravidade da doença, como descrito anteriormente 5,7. <ol><li> Atribuir escores clínicos como no passo 6.1 8. </li><li> Medir o comprimento do cólon e do peso (Figura 2C). </li><li> Realizar análise histológica de inflamação5. </li><li> Determine a expressão das citocinas espontânea em intestinais culturas de explantes tecido 9, linfa mesentéricos 10, e / ou soro 11. <ol><li> Resumidamente, para culturas de explantes 9, remover dois pontos após o sacrifício, aberta longitudinalmente, e limpo, com PBS. Incubar dois pontos, num agitador orbital a 250 rpm, em Meio Completo, durante 30 min à temperatura ambiente. </li><li> Picar o tecido em pequenos pedaços e incubados a 37 ° C em Meio Completo, durante 24 h. Recolhe-se o sobrenadante e utilizar para a quantificação de citocinas por 100 mg de tecido por ELISA. </li></ol></li><li> Realizar ex vivo caracterização de fenótipos de células T e / ou função 10. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Aqui nós descrevemos um protocolo passo-a-passo induzindo a inflamação do cólon em camundongos por transferência adotiva de células CD4 + T + CD45RB em ratos imunodeficientes. Usamos baços C57BL / 6 doadores e RAG1 singeneico - / - ratinhos receptores, embora outras estirpes (por exemplo, BALB / c, 129S6 / SvEv, não-obesos diabéticos (NOD)) modelos e genéticos da imunodeficiência (por exemplo, SCID, RAG2 - / -) também pode ser ut...

The inflammatory bowel diseases (IBD) Crohn&#8217;s disease and ulcerative colitis result from an incompletely defined and complex interaction between host immune responses, genetic susceptibility, environmental factors, and the enteric luminal contents1. Recent genome-wide association studies report associations between immune cell regulatory genes and IBD susceptibility2,3. Both innate and adaptive immune cell intrinsic genes are represented in these studies, indicating a central role for these cell populations in IBD pathogenesis.
There currently exist more than 50 animal models of human IBD. While no one model perfectly phenocopies human IBD, many are useful for studying various aspects of human disease, including disease onset and progression and the wound-healing response. In the method described here, intestinal inflammation is initiated with syngeneic splenic CD4+CD45RBhigh T cell adoptive transfer into T and B cell deficient recipient mice4. The CD4+CD45RBhigh T cell population contains mainly na&#239;ve T cells primed for activation that are capable of inducing chronic small bowel and colonic inflammation. This method allows the researcher to modify key experimental variables, including both innate and adaptive immune cell populations, to answer biologically relevant questions relating to disease pathogenesis. Additionally, this method provides precise initiation of disease onset and a well-characterized experimental time course. This permits the kinetic study of clinical features of disease progression in mice. Intestinal inflammation induced by this method shares many features with human IBD, including chronic large and small bowel transmural inflammation, pathogenesis driven by cytokines such as TNF and IL-12, and systemic symptoms such as wasting5. Thus, it is an ideal model system for studying the pathogenesis of human IBD.
The method here describes in detail the protocol for inducing experimental colitis by adoptive transfer of CD4+CD45RBhigh T cells into Rag1-/- mice. We discuss key technical steps, expected results, optimization, and trouble-shooting. We address the required elements for the successful development of this murine model of intestinal inflammation for research purposes.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Name of Reagent/ Equipment</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments/Description</td>
		</tr>
		<tr >
			<td >10x PBS</td>
			<td >Gibco</td>
			<td >14200075</td>
			<td></td>
		</tr>
		<tr >
			<td >12x75mm round-bottom tube</td>
			<td >Falcon</td>
			<td >352052</td>
			<td></td>
		</tr>
		<tr >
			<td >15 ml conical</td>
			<td >Corning</td>
			<td >430790</td>
			<td></td>
		</tr>
		<tr >
			<td >26g x 3/8 Needle</td>
			<td >BD Biosciences</td>
			<td >305110</td>
			<td></td>
		</tr>
		<tr >
			<td >50 ml conical</td>
			<td >Corning</td>
			<td >430828</td>
			<td></td>
		</tr>
		<tr >
			<td >70 um Cell Strainer</td>
			<td >Fisherbrand</td>
			<td >22363548</td>
			<td></td>
		</tr>
		<tr >
			<td >BD IMagnet</td>
			<td >BD Biosciences</td>
			<td >552311</td>
			<td></td>
		</tr>
		<tr >
			<td >&#946;-mercaptoethanol</td>
			<td >Thermo Scientific</td>
			<td >35602</td>
			<td></td>
		</tr>
		<tr >
			<td >CD4-FITC IgG2b</td>
			<td >eBioscience</td>
			<td >11-0041</td>
			<td></td>
		</tr>
		<tr >
			<td >CD45RB-PE IgG2a</td>
			<td >BD Pharminogen</td>
			<td >553101</td>
			<td></td>
		</tr>
		<tr >
			<td >Complete Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 10% FBS, 0.0004% &#946;-ME</td>
		</tr>
		<tr >
			<td >FACS tube + strainer</td>
			<td >BD Falcon</td>
			<td >352235</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Microscope Slides</td>
			<td >Fisherbrand</td>
			<td >12550A3</td>
			<td></td>
		</tr>
		<tr >
			<td >Heat-inactivated FBS</td>
			<td >Gemini</td>
			<td >100-106</td>
			<td></td>
		</tr>
		<tr >
			<td >Labeling Buffer</td>
			<td ></td>
			<td ></td>
			<td >1x PBS, 0.5% BSA, 2 mM EDTA</td>
		</tr>
		<tr >
			<td >Lysis Buffer</td>
			<td ></td>
			<td ></td>
			<td >0.08% NH4Cl, 0.1% KHCO3, 1 mM EDTA</td>
		</tr>
		<tr >
			<td >MoFlo XDP</td>
			<td >Beckman Coulter</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse CD4 T lymphocyte Enrichment Set - DM</td>
			<td >BD Biosciences</td>
			<td >558131</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2a-PE</td>
			<td >BD Pharminogen</td>
			<td >553457</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2b-FITC</td>
			<td >eBioscience</td>
			<td >11-4732</td>
			<td></td>
		</tr>
		<tr >
			<td >Pasteur pipet</td>
			<td >Fisherbrand</td>
			<td >13-678-20D</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin Solution, 100X</td>
			<td >Corning Cellgro</td>
			<td >30-002-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Petri Dish</td>
			<td >Fisherbrand</td>
			<td >875713</td>
			<td></td>
		</tr>
		<tr >
			<td >Pure Ethanol 200 Proof</td>
			<td >Decon Labs</td>
			<td >2705-HC</td>
			<td></td>
		</tr>
		<tr >
			<td >RPMI-1640</td>
			<td >Gibco</td>
			<td >11-875-093</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringe</td>
			<td >BD Biosciences</td>
			<td >309597</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypan blue</td>
			<td >Corning Cellgro</td>
			<td >25-900-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Wash Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 0.0004% &#946;-ME</td>
		</tr>
	</tbody>
</table>

induction of murine intestinal inflammation by adoptive transfer of effector cd4+cd45rbhigh t cells into immunodeficient mice

Existem muitos modelos diferentes de animais disponíveis para estudar a patogênese de doenças humanas inflamatórias intestinais (DII), cada um com suas próprias vantagens e desvantagens. Descrevemos aqui um modelo de colite experimental que é iniciada por transferência adoptiva de células de baço singeneicas CD4 + CD45RB high T em ratinhos deficientes em receptores de células T e B. A população de células T CD4 + elevada CD45RB que consiste em grande parte de células efectoras naive é capaz de induzir a inflamação intestinal crónica, assemelhando-se intimamente os aspectos chave da DII humana. Este método pode ser manipulado para estudar os aspectos do aparecimento e progressão da doença. Além disso, ele pode ser utilizado para estudar a função de inata e adaptativa, e as populações de células imunitárias reguladoras, e o papel da exposição ambiental, ou seja, a microbiota, na inflamação intestinal. Neste artigo, ilustram a metodologia para a indução de colite com um protocolo passo-a-passo. Este includes um vídeo de demonstração de aspectos técnicos essenciais necessários para desenvolver com sucesso este modelo murino de colite experimental para fins de pesquisa.

Aproximadamente 10 x 10 6 de células T CD4 + CD45RB high T de baços de 10 adultos C57BL / 6 murganhos dadores são seguramente isolado. Este número variará, dependendo da idade e estirpe do rato dador e a proficiência do investigador. Quando 4 x 10 5 C57BL / 6 células CD4 + T CD45RB alta são transferidos para C57BL / 6 RAG1 - / - ratinhos receptores, sinais clínicos da doença surgir em torno semana 5 pós-repletion ou antes, s...

Watch this Scientific Journal Video about Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice at JoVE.com

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Indução de Murino inflamação intestinal por transferência adotiva de Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; Células T em imunodeficientes Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

induction-murine-intestinal-inflammation-adoptive-transfer-effector

Research

JoVE Journal

Immunology and Infection

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

16.2K visualizações.  University of North Carolina at Chapel Hill. Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Vídeo: Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Metástase experimental e CTL Adoptive Transferência Modelo rato Imunoterapia

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Imunologia e Infecção

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Análise por Citometria de fluxo das células imunológicas Dentro aortas murino

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

Accelerated Diabetes Tipo 1 Indução em Ratos por transferência adotiva de diabetogênicos TCD4 +

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

Avaliando o Desenvolvimento da murino células dendríticas plasmocitóides em placas de Peyer Usando transferência adoptiva de Progenitores hematopoiéticos

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Geração de células T para CAR adoptivo Therapy no Contexto do Glioblastoma Padrão de Atendimento

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Análises de<em&gt; Yersinia enterocolitica</em&gt; Effector translocação em células hospedeiras Usando Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

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

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

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 ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Um método não-invasivo e tecnicamente não-intensivo para indução e fenotipagem of Experimental pneumonia bacteriana em ratos

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

Indução de paralisia e lesão do sistema Visual em camundongos por específicos de células T para Neuromielite Óptica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Estereotáxica transferência adotiva de citotóxicas células imunes em modelos murino de humanos ortotópico Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...