Este trabalho foi financiado por subvenções dos Institutos Nacionais de Saúde e DK074656 AI56374 (CEM), bem como o Juvenile Diabetes Research Foundation.

1. Preparação de células <ol><li> Cultura de células alvo: Cultura do mouse linhas de células beta, NIT-1 e NIT-4, foi um 1,2 descrito anteriormente em DMEM suplementado com 10% de SFB, BSA 0,02%, não-essenciais Aminoácidos, 15mM HEPES, 16.5mm Glocose e penicilina / estreptomicina. Por 51 Cr rotulagem, as células de semente em um flat-bottom de 96 poços na placa de cultura 5X10 4 células / poço em 200 mL meio de cultura. Para experimentos de microscopia, as células de semente em um oito bem-Lab-Tak II chambered lamela em 5x10 4 por câmara em 500 mL meio de cultura, e deixe crescer por 48 horas antes de usar nos experimentos de citotoxicidade. </li><li> Controle T cultura linha celular: Use a linha de células Jurkat (ATCC, CD3 + linha de células de linfócitos humanos) como controle negativo. Cultura de células Jurkat em RPMI1640 suplementado com 10% de SFB, 2 mM L-glutamina, bicarbonato de sódio 1,5 g / L, 10 mM HEPES e 1,0 mM piruvato de sódio. </li></ol> 2. Coleta e ativação de efetores autoreactive células T CD8 + NOD.Cg-RAG1 tm1Mom Tg (TcraAI4) 1Dvs/DvsJ [NOD-AI4a] e NOD.Cg-RAG1 tm1Mom Tg (TcrbAI4) 1Dvs/DvsJ [NOD-AI4b] foram adquiridos do Laboratório Jackson (Bar Harbor, ME) e criados em nossa unidade de mouse. Progênie híbrida F1 a partir de acasalamentos de NOD-AI4a com NOD-AI4b [NOD-AI4a / b] desenvolver diabetes em 3-5 semanas de idade. Todos os animais foram alojados em patógenos específicos livre de instalações e aprovadas pelo Atendimento a instituição do Comitê Animal e Uso. <ol><li> Euthanize 3-4 semanas de idade NOD-AI4a / b camundongos usando CO 2. </li><li> Coletar baços de camundongos. Camundongos sacrificados são preparados com spray de etanol e abriu sob o capô cirúrgico. Baços são removidos e colocados em gelo frio tampão HBSS imediatamente. </li><li> Coletar células do baço usando um homogeneizador de vidro 7 mL. Dois a três baços cada são colocados em 5 mL de HBSS frio e homogeneizado com um 7 mL de vidro homogeneizador. Homogeneizado é coletado e centrifugado a 1200rpm por 5 minutos a 4 ° C usando um Sorvall RT7 centrífuga +. Pellets celulares são recolhidos. </li><li> Remover as células vermelhas do sangue usando o tratamento solução hipotônica. Pelotas são tratados com 5 mL de solução hipotônica / baço em gelo durante 5 minutos, em seguida neutralizada pela adição de igual volume de HBSS. Coletar células por centrifugação, como acima, e ressuspender em 50 HBSS mL. Passe as células em suspensão através de peneira de células e contagem utilizando um hemocitômetro com coloração TrypanBlue. </li><li> Ativam as células. Ressuspender as células em 5x10 6 mL / e ativar usando uma cultura 3 dias com 0,1 mM AI4 mimotope (YFIENYLEL sequência de aminoácidos) e 25 U / mL IL-2 em RPMI1640 suplementado com 10% de SFB, 2 mM L-glutamina, 1,5 g / L de bicarbonato de sódio, 10 mM HEPES e 1,0 mM piruvato de sódio. </li></ol> 3. 51 ensaio de liberação de Cr para examinar CML <ol><li> Células-alvo etiqueta: Add 51 Cr para atingir as células em 1 μCi / poço e cultura para 3 horas, e lavar 3 vezes com meio de cultura. </li><li> Suspender células efetoras em meio RPMI 1640, como mencionado no passo 2,5 para que o volume final adicionados a cada poço é de 200 mL. </li><li> Após a lavagem final das células-alvo rotulado, adicione ativação de células efetoras para as culturas de células-alvo em efetoras desejado para o destino (E: T) rácios. Nós usamos geralmente E: relações T em 0.4:1, 1:1, 2:1, 5:1, 10:1, 20:1 e 50:1. </li><li> Adicionar novas modificada RPMI 1640 (veja o passo 2.5) a 6 poços de células-alvo para atuar como um controle de lise espontânea. </li><li> Co-cultura de células efetoras e meta para 16 horas. </li><li> Recolher o sobrenadante em tubos de vidro 6x50 milímetros Lime </li><li> Lisar células adicionando 100μl SDS 2% e pipetagem várias vezes suavemente. </li><li> Coletar lisado celular em outro conjunto de 6x50 milímetros tubos de vidro Lime. </li><li> Lavar uma vez com poços limpa H 2 O e adicione a lavagem para os tubos lisado celular. </li><li> Contar os sobrenadantes e lisados ​​celulares usando um contador gama. </li><li> Calcular a lise específica da seguinte forma: <img src="files/ftp_upload/2724/2724eq1.jpg" alt="Equação 1" /></li></ol> 4. Coloração de células para imagens de células vivas Entre os mais comumente utilizados corantes potencial de membrana mitocondrial, TMRM exibe a toxicidade mitocondrial, pelo menos. 3 Por isso, escolhemos TMRM para estes estudos de imagem de células vivas. <ol><li> Prepare uma solução de 40 mM TMRM estoque em DMSO e armazenar a -20 ° C. Faça uma [25 nM] solução de trabalho fresca em vermelho de fenol livre de DMEM com todos os suplementos para NIT-1 cultura de células 1. </li><li> Mancha-alvo NIT-1 com células TMRM 25 nM por 30 min a 37 ° C. </li><li> Substituir a mídia com a mídia fresco contendo 5 nM TMRM para manter a distribuição de equilíbrio dos 4 corante. </li><li> Contar efetoras ativadas AI4 células T usando um hematocytometer. </li><li> Mancha efetoras ativadas AI4 células T com 1 ml / mL para Picogreen5 minutos à temperatura ambiente e lave com vermelho de fenol livre de meio de cultura 3 vezes. </li></ol> 5. Avaliação de linfócitos T mediada por morte de células beta usando imagens de células vivas <ol><li> Montar a lamínula chambered com células NIT manchada no palco de uma Zeiss LSM 510 microscópio confocal. Remova a guia com um dremmel e gás esterilizar o vidro com óxido de câmaras Etheline. O palco é equipado com uma câmara de imagens ao vivo de células que tem temperatura controlada a 37 ° C e 5% CO 2 com a umidade. </li><li> Adicionar ativado e corados células T para câmaras designados ao E diferente: relações T. </li><li> Adicionado mesmo número de células Jurkat manchada para as câmaras de controle. </li></ol> 6. Obtenção de imagens de células vivas O microscópio é equipado com um palco motorizado que nos permitiu adquirir imagens repetidas vezes em diferentes locais das câmaras iguais ou diferentes automaticamente ao longo do tempo do experimento. <ol><li> Usando uma lente 63x de petróleo objetivo, tirar fotos em um intervalo de três minutos cada para um total de 300 quadros por localização. </li><li> Detectar coloração TMRM usando um comprimento de onda de excitação de 543 nm e conjunto de filtros para emissão de 565-619 nm </li><li> Detectar Picogreen coloração com uma excitação 488 nm e um conjunto de filtros para emissão de 500-630 nm. O vídeo foi criado usando software Zeiss LSM. </li></ol> 7. Conversão de vídeo para publicação <ol><li> Usando o software LSM Zeiss, criar um arquivo de filme não compactado em um formato AVI. </li><li> Para converter o vídeo para o formato publicado, importe o vídeo para o pacote de software FIJI ( <a href="http://pacific.mpi-cbg.de/wiki/index.php/Fiji">http://pacific.mpi-cbg.de/wiki/index.php/Fiji</a> ), um ImageJ ( <a href="http://rsb.info.nih.gov/ij/">http://rsb.info. nih.gov / ij /</a> ) de distribuição, usando o plugin Bioformats de locos ( <a href="http://www.loci.wisc.edu/software/bio-formats">http://www.loci.wisc.edu/software/bio-formats</a> ). </li><li> Ajustar as configurações de brilho e contraste para equilibrar os sinais verde e vermelho ea taxa de quadros foi definido como 2 frames / segundo usando a animação do menu de opções. </li><li> Recortar o vídeo para 512 x 512 pixels e exportar como um arquivo AVI sem compressão. </li><li> Abrir este arquivo AVI no QuickTime 7 software (computador Apple, Cupertino, CA) e comprimir e de exportação no formato aberto de arquivo de vídeo H264/MPEG-4 a 1200 kbs / segundo. </li></ol> 8. Resultados representativos: <ol><li> Um resultado representativo de 51 ensaio CML Cr. A Figura 1 mostra um resultado representativo de um ensaio onde CML lise por cento específicos (eixo Y) aumenta à medida que o Effector: relação Target (eixo X) aumenta. Células-alvo são ilhotas primária isolada a partir de NOD imunodeficientes RAG1-/ -. Ratos e rotulado com 51 Cr. Esplenócitos dos NOD.AI4α / β camundongos transgênicos são isoladas e ativados como descrito na etapa 2 acima. Metas e efetores foram co-cultivados por 16 horas. Lise por cento específico é calculado conforme descrito no passo 3.11. </li><li> Dois representante (positivo e negativo) resulta de um T-mediada por células beta dissipação de célula de membrana mitocondrial em potencial são apresentados. Células são coradas e reações são registradas como descrito nas etapas 4, 5 e 6 acima, arquivos de vídeo são criados como no passo 7. Vídeo 1: O mouse linhagem de células beta NIT-1 foi manchado o potencial de membrana mitocondrial TMRM corante (vermelho). Ativado, autoreactive CD8 + células T AI4 (verde manchado com Picogreen) foram co-cultivados com essas NIT-1 células em uma razão E: T de 50:1. NIT-1 potencial de membrana mitocondrial dissipada gradualmente durante uma duração de 400 minutos, mas apenas nos clusters que foram interagindo com células T AI4. Vídeo 2: Como um experimento de controle, NIT-1 as células foram coradas com TMRM e co-cultivados com células Jurkat Picogreen manchada em uma razão E: T de 50:1. Células Jurkat são uma linha de células T humanas que é MHC incompatíveis. NIT-1 células mantidas potencial de membrana mitocondrial durante toda a duração de 400 minutos a co-incubação período. Células Jurkat não interagir com NIT-1 clusters e, portanto, não provocar o assassinato. </li></ol><img src="files/ftp_upload/2724/2724fig1.jpg" alt="Figura 1" /> Figura 1 resultado Representante da CML usando como células-alvo primárias ilhotas isoladas de imunodeficientes NOD.Rag1-/ -. Ratos e esplenócitos de NOD.AI4α / β camundongos transgênicos. Metas e efetores foram co-cultivados por 16 horas. Lise por cento específica aumenta com o Effector: aumenta a proporção Target. Vídeo 1. Rato linha de células beta NIT-1 foi manchado a membrana mitocondrial potencial corante TMRM (Red). Ativado autoreactive CD8 + células T AI4 (verde manchado com Picogreen) foram co-cultivados com essas NIT-1 em células razão E: T de 50:1. NIT-1 potencial de membrana mitocondrial dissipaçãoted gradualmente durante uma duração de 400 minutos, mas apenas nos clusters que estavam interagindo com o verde AI4 células T. <a href="http://www.jove.com/files/ftp_upload/2724/video.mov">Clique aqui para assistir ao vídeo</a> . Video 2. NIT-1 as células foram coradas mesma, conforme descrito no vídeo 1 e co-cultivados com Picogreen T humanas manchada Jurkat linha celular. NIT-1 as células foram capazes de manter o potencial de membrana mitocondrial pensado a duração de 400 minutos. Células Jurkat não interagir com NIT-1 clusters e, portanto, não provocar o assassinato. <a href="http://www.jove.com/files/ftp_upload/2724/Video2.mp4">Clique aqui para assistir ao vídeo</a> .

<ol>
	<li>Hamaguchi, K., Gaskins, H. R., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIT-1,+a+pancreatic+beta-cell+line+established+from+a+transgenic+NOD/Lt+mouse.">NIT-1, a pancreatic beta-cell line established from a transgenic NOD/Lt mouse.</a> Diabetes. 40, 842-849 (1991).</li><li>Chen, J., Gusdon, A. M., Piganelli, J., Leiter, E. H., Mathews, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mt-Nd2a+modifies+resistance+against+autoimmune+Type+1+diabetes+in+NOD+mice+at+the+level+of+the+pancreatic+beta+cell.">mt-Nd2a modifies resistance against autoimmune Type 1 diabetes in NOD mice at the level of the pancreatic beta cell.</a> Diabetes. , (2010).</li><li>Scaduto, R. C., Grotyohann, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mitochondrial+membrane+potential+using+fluorescent+rhodamine+derivatives.">Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives.</a> Biophys J. 76, 469-477 (1999).</li><li>Lemasters, J. J., Ramshesh, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+mitochondrial+polarization+and+depolarization+with+cationic+fluorophores.">Imaging of mitochondrial polarization and depolarization with cationic fluorophores.</a> Methods Cell Biol. 80, 283-295 (2007).</li><li>Abel, M., Krokowski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+immune-mediated+(type+1)+diabetes+mellitus:+potential+for+immunotherapy.">Pathophysiology of immune-mediated (type 1) diabetes mellitus: potential for immunotherapy.</a> BioDrugs. 15, 291-301 (2001).</li><li>Mathews, C. E., Graser, R. T., Savinov, A., Serreze, D. V., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unusual+resistance+of+ALR/Lt+mouse+beta+cells+to+autoimmune+destruction:+role+for+beta+cell-expressed+resistance+determinants.">Unusual resistance of ALR/Lt mouse beta cells to autoimmune destruction: role for beta cell-expressed resistance determinants.</a> Proc Natl Acad Sci U S A. , 235-240 (2001).</li><li>Szabadkai, G., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+mediated+cell+death+in+diabetes.">Mitochondria mediated cell death in diabetes.</a> Apoptosis. 14, 1405-1423 (2009).</li><li>Ly, J. D., Grubb, D. R., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+membrane+potential+(deltapsi(m))+in+apoptosis;+an+update.">The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update.</a> Apoptosis. 8, 115-128 (2003).</li><li>Dudek, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+T-cells+from+T-cell+receptor+transgenic+NOD8.3+mice+destroy+beta-cells+via+the+perforin+and+Fas+pathways.">Cytotoxic T-cells from T-cell receptor transgenic NOD8.3 mice destroy beta-cells via the perforin and Fas pathways.</a> Diabetes. 55, 2412-2418 (2006).</li><li>Pakala, S. V., Chivetta, M., Kelly, C. B., Katz, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+autoimmune+diabetes+the+transition+from+benign+to+pernicious+insulitis+requires+an+islet+cell+response+to+tumor+necrosis+factor+alpha.">In autoimmune diabetes the transition from benign to pernicious insulitis requires an islet cell response to tumor necrosis factor alpha.</a> J Exp Med. 189, 1053-1062 (1999).</li></ol>

Experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pela Universidade da Flórida Animal Care Institucional e Comitê de uso.

Citotóxica de células T morte celular mediada beta está a fisiopatologia das principais DM1 5. CML ensaios utilizando 51 Cr lançamento nos permitem estudar o grau de meta-efetoras de resposta 6. No entanto, o processo detalhado e os caminhos da morte das células T mediada por células beta ainda não é totalmente compreendido. Uma vez que as mitocôndrias são críticos para a função das células beta ea morte 7, centrada em mudanças mitocondrial durante a CML visual....

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Cr-51 </td><td> Perkin Elmer </td><td> NEZ030500IMC </td><td></td></tr><tr><td> PBS </td><td> Cellgro </td><td> 21-040-60 </td><td></td></tr><tr><td> Tetrametil Rodamina Methyl Ester (TMRM) </td><td> Invitrogen </td><td> T668 </td><td></td></tr><tr><td> Picogreen </td><td> Invitrogen </td><td> P7581 </td><td></td></tr><tr><td> AI4 mimotope </td><td> mimotopes.com </td><td> seqüência de aminoácidos: YFIENYLEL </td><td></td></tr><tr><td> IL-2 </td><td> R &amp; D </td><td> 485-MI </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11885 </td><td></td></tr><tr><td> FBS </td><td> HyClone </td><td> SH30071.03 </td><td></td></tr><tr><td> Albumina de soro bovino </td><td> Sigma </td><td> A8412 </td><td></td></tr><tr><td> HEPES </td><td> Cellgro </td><td> 25-060-Cl </td><td></td></tr><tr><td> MEM Non-Aminoácidos Essenciais </td><td> GIBCO </td><td> 11140 </td><td></td></tr><tr><td> Penicilina / estreptomicina </td><td> Gêmeos </td><td> 400-109 </td><td></td></tr><tr><td> Vermelho de fenol livre de DMEM </td><td> Sigma </td><td> D5303 </td><td></td></tr><tr><td> Incubadora de CO2 </td><td> Thermo Fisher </td><td> HeraCell 150i </td><td> Mantido estéril para cultura de células </td></tr><tr><td> Cabines de segurança biológica </td><td> Thermo Fisher </td><td> Forma 1440 </td><td></td></tr><tr><td> Descartáveis ​​tubos de cultura, 6x50 milímetros de vidro Lime </td><td> Pescador </td><td> 14-958-A </td><td></td></tr><tr><td> Contador gama </td><td> Perkin Elmer </td><td> Assistente de contador gama automático 1470 </td><td></td></tr><tr><td> Microscópio confocal </td><td> Zeiss </td><td> LSM 510 Meta Confocal </td><td> Com estágio motorizado e câmara de células vivas </td></tr><tr><td> Pipeta (1ml, 200μl, 20μl, 10μl, 2μl) </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> Tubos (Amber) </td><td> Pescador </td><td> 50819772 </td><td></td></tr><tr><td> Centrifugador </td><td> Sorvall Legend RT + </td><td> 75004377 </td><td></td></tr><tr><td> Hemocitômetro </td><td> Fisher Scientific </td><td> 267110 </td><td></td></tr><tr><td> Microscópio na posição vertical </td><td> Zeiss </td><td> Axioskop40 </td><td></td></tr><tr><td> NOD.Cg-RAG1 tm1Mom Tg (TcraAI4) 1Dvs/DvsJ Ratos </td><td> O Laboratório de Jackson </td><td> 004347 </td><td></td></tr><tr><td> NOD.Cg-RAG1 tm1Mom Tg (TcrbAI4) 1Dvs/DvsJ Ratos </td><td> O Laboratório de Jackson </td><td> 004348 </td><td></td></tr><tr><td> NOD-AI4α / β camundongos F1 </td><td> N / A </td><td> N / A </td><td> UF criados em instalações para animais </td></tr></tbody></table>

methods to assess beta cell death mediated by cytotoxic t lymphocytes

Diabetes tipo 1 (DM1) é uma doença mediada por células T auto-imunes. Durante a patogênese, os pacientes tornam-se progressivamente mais insulinopenic como a produção de insulina está perdido, presumivelmente, o que resulta da destruição das células beta pancreáticas pelas células T. Compreensão dos mecanismos de morte das células beta durante o desenvolvimento do DM1 irá fornecer informações para gerar uma cura eficaz para esta doença. Mediada por células linfocitotoxicidade (CML) ensaios têm historicamente utilizado o Chromium radionuclídeos 51 (51 Cr) para rotular as células-alvo. Estas metas são, em seguida, expostos a células efetoras e da liberação de 51 Cr a partir de células-alvo é lido como uma indicação de linfócitos mediada por morte celular. Inibidores de resultado morte celular na liberação diminuída de 51 Cr. Como células efetoras, usamos uma população autoreactive ativado clonal de linfócitos CD8 + T citotóxicas (CTL) isolado de um camundongo transgênico estoque para ambas as cadeias alfa e beta do receptor de células T AI4 (TCR). Ativado AI4 células T foram co-cultivados com células-alvo 51 Cr rotulados NIT por 16 horas, a liberação de 51 Cr foi gravado para calcular a lise específica Mitocôndrias participar em muitos eventos fisiológicos importantes, como a produção de energia, regulação da transdução de sinalização e apoptose. O estudo da célula beta mitocondrial alterações funcionais durante o desenvolvimento do DM1 é uma área nova de pesquisa. Usando o potencial de membrana mitocondrial corante Rodamina Tetrametil Methyl Ester (TMRM) e imagens de células microscópicas confocal vivemos, nós monitorados potencial de membrana mitocondrial ao longo do tempo na linha da célula beta NIT-1. Para exames de imagem, AI4 células T efectoras foram marcadas com o corante fluorescente coloração nuclear Picogreen. NIT-1 e células T foram co-cultivados em câmaras lamínula e montado sobre o palco microscópio equipado com uma câmara de células vivas, a 37 ° C, com 5% de CO 2, e umidificado. Durante esses experimentos imagens foram tiradas de cada cluster a cada 3 minutos para 400 minutos. Mais de um curso de 400 minutos, observou-se a dissipação do potencial de membrana mitocondrial em NIT-1 grupos de células, onde AI4 células T foram anexados. No experimento controle simultâneo onde NIT-1 as células foram co-cultivados com MHC desencontradas células Jurkat linfócitos, potencial de membrana mitocondrial permaneceu intacta. Esta técnica pode ser usada para observar em tempo real as mudanças no potencial de membrana mitocondrial em células sob ataque de linfócitos citotóxicos, citocinas, ou outros reagentes citotóxicos.

Watch this Scientific Journal Video about Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes at JoVE.com

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Mediada por células-linfocitotoxicidade (CML) os ensaios podem ser usados ​​para testar as respostas auto-reativos e mecanismos de estudo de morte celular in vitro. No entanto, usando live-célula confocal técnicas de imagem microscópica com corantes fluorescentes, do tipo e da cinética de morte celular, bem como as vias utilizadas podem ser estudados em maior detalhe.

Métodos para avaliar Mediated Beta Morte celular por linfócitos T citotóxicos

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

methods-to-assess-beta-cell-death-mediated-by-cytotoxic-t-lymphocytes

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JoVE Journal

Immunology and Infection

13.3K visualizações.  University of Florida. Mediada por células-linfocitotoxicidade (CML) os ensaios podem ser usados ​​para testar as respostas auto-reativos e mecanismos de estudo de morte celular in vitro. No entanto, usando live-célula confocal técnicas de imagem microscópica com corantes fluorescentes, do tipo e da cinética de morte celular, bem como as vias utilizadas podem ser estudados em maior detalhe.

Vídeo: Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Expandindo linfócitos T citotóxicos do sangue do cordão umbilical que citomegalovírus Target, vírus de Epstein-Barr, e Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Imunologia e Infecção

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

Determinando a atividade citotóxica Optimal específicos de células CD8 Her2neu Humanos T, comparando a Cr51 Ensaio de Libertação do Sistema xCELLigence

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Diferenciação de células Dirigido tronco pluripotentes induzidas no sentido de Linfócitos T

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

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 ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Baseada em células de Citometria de Fluxo Ensaio medir citotóxicos Atividade

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

Radial Mobilidade e função citotóxica de Retroviral Replicando Vector transduzidas, Alloresponsive não aderentes Linfócitos T

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

Difusão molecular em membranas plasmáticas de linfócitos primários medida por fluorescência Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

Quantificação rápida de blastogénese induzida por Mitogénio em linfócitos T para identificar drogas imunomoduladoras

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Um método para avaliar funções efetoras mediada por Fc induzidas por anticorpos específicos de hemaglutinina de gripe

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.

Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

We quantify epidermal cell death in frogs with chytridiomycosis using two methods. First, we use terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ histology to determine differences between clinically infected and uninfected animals. Second, we conduct a time series analysis of apoptosis over infection using a caspase 3/7 protein analysis.

Usando o Terminal Transferase-mediada dUTP Nick End-rotulagem (TUNEL) e Caspase 3/7 ensaios a morte de células epidérmicas medida em sapos com quitridiomicose

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is ...