Este trabalho é apoiado pelo NIH PBS (R21 AI095484). Agradecemos NHI tetramer instalação do núcleo para CD1d tetrâmero.

1. Preparação de Instrumentos, Meios de Cultura e Reagentes <ol><li> Instrumentos: Preparar os reagentes em condições estéreis e usar em uma capela de fluxo laminar. </li><li> Prepare 1 litro de meio de cultura RPMI tecidos, adicionam-se 5% de soro fetal bovino, 2,1 g de bicarbonato, 10 ml de glutamato de 100 vezes, 10 mg de HEPES, piruvato de sódio 1 mg e 10 ml de 100x de penicilina / estreptomicina. Nota: o meio de cultura de tecidos recomendados, RPMI, é intercambiável com DMEM. </li><li> Preparar a solução de 5% de colagenase &quot;D&quot; (5 ml de colagenase &quot;D&quot; a 9 ml de meio de cultura de tecido). </li><li> Adicione uma solução de Percoll a três concentrações diferentes: 100%, 80% e 40%. As seguintes concentrações são calculados para um amostra. Manter as soluções à temperatura ambiente. <ol><li> Solução de Percoll: adicionar 9 ml de Percoll (não diluído de solução estoque); adicionar 1 ml de PBS 10x. </li><li> Solução de Percoll: adicionar 8 mL de Percoll a 100%; adicionar 2 ml de PBS 1x. </li><li> Solução de Percoll:adicionar 4 ml de Percoll a 80%; adicionar 4 mL de PBS 1x. </li></ol></li><li> Adicione 1 L de células activadas por fluorescência tampão (FACS); adicionar 5 ml de 10% BSA (0,1% BSA), azida de sódio a 1 ml a 10% (0,1%), 2 ml agente EDTA, fazer até 1.000 ml com PBS 1x. </li></ol> 2. Preparação de rim Digestão <ol><li> Tendo adquirido qualquer aprovação institucional necessário, sacrificar e desangrar os ratos com métodos padronizados e seguindo as normas vigentes e orientações de cuidados de animais. <ol><li> Anestesiar o mouse com pentobarbital (0,07 mg / kg). </li><li> Vá para a sangria. </li><li> Posicione o mouse em decúbito dorsal sobre uma mesa cirúrgica. </li><li> Pulverizar a área abdominal com etanol a 70%. </li></ol></li><li> Adicionar 10 ml de solução de colagenase D para a placa de Petri. </li><li> Retire e decapsulate ambos os rins de cada rato. <ol><li> Abrir a cavidade abdomimal fazendo uma incisão na linha média abdominal através da pele e do peritoneu do esternoo púbis. </li><li> Expor o rim esquerdo movendo o intestino lateralmente para o lado. </li><li> Cuidadosamente deixou separar a cápsula com a pinça. Nota: A cápsula aparece como uma camada transparente em torno do rim e parcialmente coberta por tecido adiposo perirrenal. </li><li> Remover o rim esquerdo cortando os vasos do pedículo usando uma tesoura cirúrgica. </li><li> Repita o mesmo procedimento para o rim direito. Nota: O rim direito é mais cranial e mais perto da linha média (pedicelos são mais curtas). </li></ol></li><li> Coloque o rim para a placa de Petri. </li><li> Cortar o tecido renal em pedaços pequenos (1-2 mm de dimensão) usando uma lâmina de moldagem de metal regular e colocar as peças na solução de colagenase durante 30-45 minutos na incubadora a 37 ° C (Figura 1). </li></ol> 3. Preparação de células mononucleares do rim (KMNC) da Digestão rim <ol><li> Se obter uma suspensão de células do rimdigestão por disrupção mecânica do tecido com um filtro (70 mm) e ressuspender em 25 ml de meio de cultura de tecidos e misturar. </li><li> Centrifugar a 400 xg durante 10 min a 4 ° C para sedimentar a suspensão de células. </li><li> Re-suspender o sedimento em 4 ml de solução de Percoll a 40% e suavemente sobrepor em 4 ml de uma solução de Percoll a 80% com uma pipeta de transferência de plástico, muito lentamente e cuidadosamente. Isto irá resultar em duas fases claramente separadas por uma camada translúcida. Na parte superior vai ser uma camada amarela correspondente aos lípidos. </li><li> Centrifugar a 1.500 xg durante 30 minutos à temperatura ambiente com a centrífuga no freio modo. </li><li> Remover por aspiração a camada superior amarela e espessa (cerca de 1-2 ml). </li><li> Recolhe apenas uma ligeira camada translúcida esbranquiçado a partir da interface das duas fases, com uma pipeta de transferência. Transferir muito cuidadosamente esta suspensão em um tubo de 15 ml contendo 2 ml de meio de cultura de tecido e, em seguida, adicionar 13 ml de meio para perfazer um volume total de15 ml. </li><li> Centrifugar a 400 xg durante 10 min a 4 ° C. </li><li> Ressuspender as amostras em 1 ml de meio. </li><li> Contar o número de KMNC usando exclusão com azul de tripano em um hemocitómetro e exprimir os resultados como o número de KMNC por ml por rim. </li><li> Lavar uma vez como acima. </li></ol> 4. Isolamento de hematopoiéticas (CD45 +) Células de KMNC (Passo I) <ol><li> Ressuspender as células até 10 7 em 90 ul de tampão de corrida. </li><li> Adicionar 10 ul de microesferas de CD45 e incubar durante 15 min a 4 ° C. </li><li> Adicionar 1 ml de tampão de corrida para parar a reacção. Centrifugar a 400 xg durante 10 min a 4 ° C e ressuspender em 500 ul de tampão de corrida. </li><li> Prepare a coluna magnética, colocando no ímã e enxágüe com 3 ml de tampão de corrida. </li><li> Passar a suspensão de células através de uma coluna magnética e recolher as células não marcadas que passam através da coluna. </li><li> Lave as células três vezes com 3 ml de runnção tampão. Em seguida, coletar o efluente. </li><li> Remova a coluna do separador e pipeta de 5 ml de tampão de corrida para as colunas e expulsar a fração rotulado imediatamente. Esta fracção contém as células CD45 +. Contar as células. </li><li> Para calcular o número absoluto de células T diferentes subconjuntos em rins multiplicar o número total de KMNC pela percentagem de células positivas determinada por citometria de fluxo (Figura 2). </li></ol> 5. Isolamento de DN celular T de CD45 + Preparação de selecção negativa (Etapa II) <ol><li> Adicionar 2,5 mL (0,5 mg / mL, 1,25 ng) de cada um dos seguintes: anticorpos biotinilados anti-CD4, anti-CD8, anti-receptor de Fc (CD16/32), anti-MHC classe II (IA b), anti- CD1d PBS-57 tetrâmero para cada 10 células hematopoiéticas 7. </li><li> Incubar as células durante 30 minutos em câmara fria. </li><li> Adicionar 1 ml de microesferas anti-biotina. </li><li> Incubar durante 30 minutos numa sala fria. </li><li> Colocar o tubo no tele ímã e proceder ao esgotamento. </li><li> Contagem de células viáveis ​​para determinar o número total de células T DN na fracção negativa, utilizando um hemocitómetro. </li><li> Verifique a pureza das células T DN com citometria de fluxo, seguindo esta estratégia de propagação: primeiro portão CD45 +, segundo portão TCRαβ +. Excluir células CD1d +. Lote T CD4 + contra CD8 + para identificar células DN T (Figura 3). </li><li> Multiplicar o número total determinado pela percentagem em pureza. Nota: O uso deste protocolo resulta em uma população de mais de 90% de células T DN puros. </li></ol>

<ol>
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Os autores não têm nada a revelar.

Há cada vez mais interesse em células T DN, uma vez que estão sendo implicados em diferentes condições patológicas, como doenças auto-imunes, câncer, tolerância enxerto e doenças primárias do rim, incluindo lesão renal aguda (LRA), glomerulonefrite 8, 13. Portanto, há necessidade de melhor compreender e caracterizar as funções fisiopatológicos de células T DN. No entanto, atualmente há uma falta de compreensão da função dessas células, em comparação com CD4 e células T CD8. Uma das pr...

A correction was made to: <a href="http://www.jove.com/video/51192/isolation-of-double-negative-t-cells-from-the-kidney">Isolation of Double Negative &#945;&#946; T Cells from the Kidney</a>.
There was an error with an author's name. The author's given name had a typo, this was corrected to:
Samatha Bandapelle
instead of:
Samantha Bandapelle

A correction was made to: Isolation of Double Negative &#945;&#946; T Cells from the Kidney.

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

Peripheral αβTCR + CD3 + CD4 - CD8 - double-negativo células (DN) T são divididos em vários subgrupos que possuem fenótipos e funções 1-4 distintas. DN células T são pouco compreendidos mas cada vez mais a ser implicadas nas respostas imunes fisiopatológicas em diferentes modelos de doença 4-6. Células DN T são um tipo de célula imunológica única que é cada vez mais implicados na regulação de várias respostas imunes e auto-imunes ea modulação da tolerância alotransplante. 4-6, 9 Eles são raros no sangue periférico e órgãos linfóides secundários (baço e linfonodos ). No entanto, eles são os principais moradores do rim e intestino epitélio normal 10-12. Muito pouco se sabe sobre sua função 7 no rim no estado estável e em condições patológicas como lesão renal aguda (LRA), associados com transpl renalformiga. Devido às funções complexas de células imunes diferentes na regulação de respostas imunes, incluindo alloresponses, a definição do papel de cada jogador é fundamental na compreensão alloresponses e concepção de novas terapias. Dado o número significativo de células T DN presentes no rim em condições fisiológicas e de doenças diferentes, as células T DN são susceptíveis de desempenhar um papel crítico na regulação das respostas imunológicas e auto-imunes em ratos e seres humanos, e alloresponses em receptores de transplante. Acumulando embora a evidência ainda espalhados implica células T DN em ambas as funções patogênicos e imunossupressores, mas é mal compreendido por que e como eles exibem uma função prejudicial ou supressiva específico e como o ambiente influencia-los. Devido à sua fraca abundância no rim, métodos melhorados de isolamento são necessárias. Não há atualmente nenhum protocolo padrão para isolamento de células T DN de the tecidos não linfóides. Nosso protocolo descreve um novo método para o isolamento de células T DN do rim; No entanto, o método também pode ser utilizado para vários tecidos não linfóides.

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			<td height="21" style="height:21px;width:439px;">Laminar flow hood&#160;</td>
			<td style="width:257px;">Baker Company, Inc</td>
			<td style="width:276px;"></td>
			<td style="width:200px;">Or equivalent equipment&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Centrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Hemocytometer</td>
			<td>Electron Microscopy Sciences</td>
			<td style="width:276px;">63514-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Atmosphere-controlled incubator</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td style="width:200px;">&#160;(37&#176;C with 5% CO2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Microscope&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Analytical flow cytometer</td>
			<td style="width:257px;">LSR II&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:439px;">Name of the reagent</td>
			<td style="width:257px;">Company&#160;</td>
			<td style="width:276px;">Catalog&#160; Number</td>
			<td style="width:200px;">Comments&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">RPMI 1640</td>
			<td style="width:257px;">Media Tech</td>
			<td style="width:276px;">10-040-CV</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Collagenase D</td>
			<td style="width:257px;">Roche</td>
			<td style="width:276px;">11088858001</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Percoll&#160;</td>
			<td style="width:257px;">GE Healthcare</td>
			<td style="width:276px;">&#160;17-0891-01</td>
			<td style="width:200px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fetal bovine serum&#160;</td>
			<td>Corning Cellgro</td>
			<td>35-011-CV</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">0.1% sodium azide (NaN3)</td>
			<td style="width:257px;">Sigma-Aldrich&#160;</td>
			<td style="width:276px;">S2002</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Buffer phosphate buffered saline (PBS)</td>
			<td style="width:257px;">Corning Cellgro</td>
			<td>21-040-CV</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">PBS 10X</td>
			<td>Mediatech</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">EDTA buffer&#160;</td>
			<td style="width:257px;">Sigma-Aldrich&#160;</td>
			<td style="width:276px;">E1161</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">LS columns&#160;</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">CD45+ microbeads</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">6.78E-51</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Biotin microbeads&#160;</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">130-090-485</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD45 (clone: 30-F11)&#160; PerCP&#160;</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td style="width:276px;">G1397</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD45 (clone: 30-F11)&#160; APC-Cy7</td>
			<td style="width:257px;">Biolegend&#160;&#160;</td>
			<td style="width:276px;">103116</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-TCR Pacific Blue&#160; (ab-chain, clone: H57-597)</td>
			<td style="width:257px;">Invitrogen</td>
			<td style="width:276px;">HM3628</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD4 PE</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td>12-0043</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD8 FITC</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td>8011-0087</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD4 biotinylated (GK1.5)</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553728</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD8 biotinylated&#160; (clone 53-6.7)</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553029</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Fc receptor (CD16/32) biotinylated</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553143</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-MHC class II (I-A d) biotinylated</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553609</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD1d PBS-57 tetramer</td>
			<td style="width:257px;">NHI tetramer&#160;</td>
			<td style="width:276px;">lote # 15621-PBS57</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:439px;">AutoMACS Running Buffer-&#160; MACS Separation Buffer&#160;</td>
			<td style="width:257px;">MIlteny I Biotec</td>
			<td style="width:276px;">130-091-221</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">C57BL/6J mice</td>
			<td>Jackson Labs</td>
			<td style="width:276px;">000664</td>
			<td>Kidneys - Lymph Nodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Petri dish&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:276px;">356517</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">70mc filter&#160;</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Plunger&#160;</td>
			<td style="width:257px;"></td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GeneMate Tubes 50 ml</td>
			<td>Bioexpress</td>
			<td>C-3394-3</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">GeneMate Tubes 15 ml</td>
			<td>Bioexpress</td>
			<td style="width:276px;">C-3394-1</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Petri Dish&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:276px;">S33580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Plate 24 wells&#160;</td>
			<td style="width:257px;">BD Biosciences</td>
			<td style="width:276px;">354723</td>
			<td style="width:200px;"></td>
		</tr>
	</tbody>
</table>

isolation of double negative &#945;&#946; t cells from the kidney

Não existe actualmente nenhum protocolo padrão para o isolamento de células T DN dos tecidos não linfóides apesar do seu envolvimento cada vez relatada em diversas respostas imunitárias. DN células T são um tipo de células imunitárias única que tem sido implicado na regulação de respostas imunes e tolerância e auto-imunes a alotransplante 1-6. Células T DN são, no entanto, raro no sangue periférico e órgãos linfóides secundários (baço e linfonodos), mas são os principais habitantes do rim normal. Muito pouco se sabe sobre sua função fisiopatológica 7, devido à sua escassez na periferia. Recentemente, descreveu uma análise fenotípica e funcional abrangente desta população no rim 8 em estado estável e durante a isquemia reperfusão. Análise da função das células T DN será consideravelmente reforçada através do desenvolvimento de um protocolo para o isolamento do rim. Aqui, descrevemos um novo protocolo que permite isolation de células altamente puros ab CD4 + CD8 + T e células T DN do rim murino. Resumidamente, podemos digerir tecido renal utilizando colagenase e isolamento de células mononucleares de rim (KMNC) por gradiente de densidade. Isto é seguido por dois passos para enriquecer as células T hematopoiéticas de 3% a 70% de KMNC. A primeira etapa consiste de uma selecção positiva de células hematopoéticas CD45 + usando um kit de isolamento. No segundo passo, as células T são DN negativamente isolado por remoção das células não desejadas usando CD4, CD8, e anticorpos monoclonais de MHC de classe II e CD1d α-GalCer tetrâmero. Esta estratégia conduz a uma população de mais do que 90% de células T DN puros. Coloração de superfície com os anticorpos acima mencionados, seguida por análise FACS é usado para confirmar a pureza.

Tipo selvagem C57BL / 6 (B6) rim contém aproximadamente 1,5-2,1 x 10 6 células mononucleares por rim. Cerca de menos de 10% são células hematopoiéticas CD45 +. Para a preparação de células mononucleares do rim (KMNC), os rins são cortadas em pedaços pequenos, como mostrado na Figura 1 seguido por digestão usando 5% de colagenase. Isto é seguido por realização de uma centrifugação de gradiente de densidade para recolher camada KMNC <stron...

Watch this Scientific Journal Video about Isolation of Double Negative Î±Î² T Cells from the Kidney at JoVE.com

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

DNabT células são raras entre as células T periféricas; no entanto, eles são abundantes em certos tecidos não linfóides. Dificuldade de isolar as células T DN a partir de tecidos não-linfóides dificulta a sua análise funcional, apesar do aumento reconhecida importância fisiopatológica. Descreve-se um novo protocolo para o isolamento de células altamente purificadas DN T de rim murino.

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 ...

isolation-of-double-negative-t-cells-from-the-kidney

Research

JoVE Journal

Immunology and Infection

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

12.1K visualizações.  Johns Hopkins University School of Medicine.  DNabT células são raras entre as células T periféricas; no entanto, eles são abundantes em certos tecidos não linfóides. Dificuldade de isolar as células T DN a partir de tecidos não-linfóides dificulta a sua análise funcional, apesar do aumento reconhecida importância fisiopatológica. Descreve-se um novo protocolo para o isolamento de células altamente purificadas DN T de rim murino. 

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

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&amp;#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 ...

Imunologia e Infecção

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

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

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

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

Isolamento e Th17 Diferenciação de Linfócitos T CD4 Naïve

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

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 ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Rato Naïve CD4<sup&gt; +</sup&gt; Isolamento de células T e<em&gt; In vitro</em&gt; Diferenciação em subpopulações de células T

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

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.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

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 ...

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

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

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

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

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

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

Isolamento e Citometria de Fluxo Analysis of Human Endocervical Gama Cells Delta T

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

Avaliação da Interface sináptica de células T humano primário do sangue periférico e tecido linfoide

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Imagem latente confocal de RNA Double-Stranded e receptores de reconhecimento padrão na infecção pelo vírus de RNA de sentido negativo

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...