Somos gratos a Alice Gaughan e Jiao Wang Jing-cujo saldo suporte técnico, contribuição intelectual, e apoio moral foram fundamentais para mover esses estudos à frente. Estes estudos foram apoiados pela concessão NIH AI036532 para GAH.

1. A irradiação letal <ol><li> Colocar-se a 10 ratinhos receptores em uma gaiola microisolator compatível com o irradiador de ser usado. </li><li> Irradiar em 2 doses iguais somando dose total (dose total = 9 cGy para receptores BALB.B). Irradiação segundo deve ser de 3 horas após a primeira. A injecção deve ocorrer entre 4-6 horas após a irradiação final. Irradiar ratos em qualquer fonte de 137 Cs ou RS2, 000 irradiador,. </li><li> Após a segunda dose de radiação ratos, armazenar em gaiola microisolator com água acidificada até o momento do transplante. </li></ol> 2. Preparação esplenócitos <ol><li> Euthanize um camundongo doador acordo com as diretrizes institucionais. Cada dador geralmente produz 75x10 6 - 125x10 6 esplenócitos. Utilizando kits de purificação de CD8, cada rato geralmente produz 6x10 6 - 12x10 6 células T CD8. </li><li> Remover o baço fazendo primeiro um verticais 2 centímetros de incisão direita 2 centímetros da linha mediana directamentesob a caixa torácica. Cortar através da pele, pele, membrana e visceral. </li><li> Remover o baço e criar suspensão de células individuais através da colocação do baço em 40 ul de tela de malha em uma placa de Petri com meio RPMI 1640 com FBS a 5%. Usar um êmbolo da seringa para quebrar o baço até ao baço inteiro foi passado através de peneiro de malha. </li><li> Repita esse processo para cada WT e L2G85.B6 doador. Recolher todos os WT suspensões de células individuais em tubo 1-50 ml, e recolher todos os L2G85.B6 suspensões de células individuais em um tubo separado 50 ml. </li><li> Centrifugar as células a 1.200 rpm durante 10 min a 4 ° C. Pelotas Ressuspender em 1640 RPMI com 5% de FBS e contagem de células. </li></ol> 3. Preparação de Medula Óssea <ol><li> Remover a pele de um membro posterior de um rato dador. </li><li> Cortar cuidadosamente como tecido muscular máximo possível de fêmur e tíbia / fíbula. </li><li> Remover membro posterior cortando fêmur afastado na articulação do quadril. Cortar pata traseira, logo abaixo da tíbia / fíbula intersecção. Todos os cortes ósseos, deve ser feita com uma tesoura resistente. </li><li> Remova cuidadosamente qualquer tecido do músculo restante. Corte-fíbula relativamente pouco medula óssea é encontrada na fíbula e não vale a pena o esforço. </li><li> Coloque membro posterior no frio 1640 RPMI processo FBS e repita 5% com a segunda parte traseira. Cada rato deve render entre 6 20x10 - 40x10 6 células da medula óssea. </li></ol> 4. Remoção de Medula Óssea <ol><li> Remover um membro posterior da mídia e coloque em placa de Petri grande com uma pequena quantidade de meios frios (~ 1 ml). </li><li> Cortar a articulação do joelho. Utilizando uma seringa (volume de ml&gt; 5), a inserção da agulha subcutâneo na tíbia e deprimir seringa até que todo o material é removido do vermelho interior da tíbia. Repita o processo com o fêmur. Descartar ossos restantes desprovidos de medula óssea. Repita o processo com o segundo membro posterior. </li><li> Criar suspensão de células individuais por meio de pipetagem contendo medula óssea em placas de Petri e grande utilização êmbolo da seringa e 40 ulmalha da tela. Pipetar única suspensão de células em 50 ml de tubo cónico e manter em gelo. </li></ol> 5. CD3 Esgotamento Há uma variedade de maneiras para empobrecem células CD3 + a partir de medula óssea. O nosso laboratório utiliza um kit fabricado por Miltenyi Biotec (CD3-biotina - 130-093-021). Empobrecem células CD3 + a partir de medula óssea após o protocolo do fabricante. O tampão para os kits de Miltenyi será daqui em diante chamado de buffer MACS (2 mM de EDTA, 0,5% de BSA em PBS, pH 7,2). <ol><li> Lave as células por centrifugação esplenócitos e células da medula óssea a 1200 rpm durante 10 min a 4 ° C as células e contagem. </li><li> Remover ALL sobrenadante. Ressuspender as células ósseas medula em 100 ul por 10 milhões de células de medula óssea em tampão MACS. </li><li> Continue com CD3 esgotamento usando fabrica protocolo &quot;. </li><li> Lavar CD3 depletados de células da medula óssea 3 vezes em PBS estéril. Contagem de CD3 empobrecido células da medula óssea e ressuspender em um volume apropriado de injectar 10 7 células. </li></ol> Há uma variedade de maneiras de purificar a partir de células T CD8 L2G85.B6 ratos. Além disso, existem várias maneiras de destroem as células T CD8 a partir de doadores de esplenócitos WT. Nosso laboratório utiliza kits de Miltenyi Biotec (CD8 exaustão - 130-049-401, CD8 purificação - 130-095-236). <ol><li> Ressuspender o sedimento em 90 WT Tampão MACS ul por 10 7 células. Continue com depleção de células T CD8 de acordo com a fabrica &quot;protocolo (CD8 exaustão - 130-049-401). </li><li> Ressuspender o sedimento em 40 L2G85.B6 Tampão MACS ul por 10 7 células. Continue com CD8 purificação de células T de acordo com a fabrica &quot;protocolo (CD8 purificação - 130-095-236). </li><li> Contar cada população de células e lava-se cada população 3 vezes com PBS estéril. Ressuspender cada população em volume adequado para injectar 18x10 6 WT (CD8 empobrecido) e esplenócitos de 2x10 6 L2G85.B6 purificado células T CD8. </li></ol>7. Preparação de injecção <ol><li> Combinar 10 7 células CD3 ósseas empobrecido medulas, 18x10 6 WT (CD8 empobrecido) e esplenócitos de 2x10 6 L2G85.B6 células T CD8 purificadas num tubo de microcentrífuga. Lavar com PBS estéril e ressuspender em 300 ul. </li><li> Injectar preparação de células para dentro da veia da cauda dos ratos receptores. As injeções devem ser feitas usando agulhas de calibre 28. </li><li> Loja ratos na gaiola microisolator com água acidificada. Camundongos pontuação diária usando pontuação sistema de pontuação GVHD desenvolvido por Cooke et al 1. </li></ol> 8. Bioluminescent imagem <ol><li> Seis dias após o transplante, injectar ratinhos receptores com 4 mg de D-luciferina. 5 min para permitir que a luciferina a reagir com a luciferase. </li><li> Anestesiar mouse na câmara de isoflurano de imageador bioluminescência e receptores de imagem por 5 minutos com binning pequeno. Isto irá criar uma imagem de alta resolução durante a coleta de tantos eventos quanto possível. </li><li> Analisar dados usando VivoSoftware de imagem. A escala da imagem pseudo-cor pode ser alterado para se obter os melhores resultados. No entanto, é imperativo que a mesma escala ser usados ​​em experimentos. </li><li> As regiões de interesse podem ser criados utilizando o software de imagem e de estar emitância luminosa pode ser quantificada por cálculo do fluxo (fotões / sec) a ser emitida a partir de cada região de interesse. </li></ol> 9. Resultados representativos Cerca de 7-10 dias após o transplante, os ratos começam a mostrar sinais clínicos da GVHD. Ratos aparecer desalinhado devido à falta de higiene. Os destinatários também vai começar a perder peso entre 7-10 dias após o transplante. Atividade e postura de ratos receptores permanecerão relativamente normal até pós-transplante aproximadamente 12-14 dias. Cumulativos pontuações GVHD irá aumentar gradualmente através do primeiro transplante pós 2-3 semanas (Figura 1A). Curso da doença é bastante variável entre os ratos, no entanto, os destinatários devem uniformemente sucumbir a DECH por d 30-40ays após o transplante (Figura 1B). A Figura 2 mostra que os ratinhos receptores foram fotografados 6 após o transplante dias. Pseudo-colorida escala mostra variando emitância luz em todo o corpo, com a maior intensidade de luz a ser emitida no baço e no intestino. CD8 acumulação de células T no intestino é consistente com descobertas anteriores 6. Ratinhos receptores podem ser colocados de volta na gaiola microisolator a ser trabalhada em um ponto de tempo posterior, ou sacrificados por ex imagiologia in vivo. <table border="1"><tbody><tr><td> Critérios </td><td> Grau 0 </td><td> Grau 1 </td><td> Grau 2 </td></tr><tr><td> Perda de peso (wkly.) </td><td> &lt;10% </td><td> &gt; 10% - &lt;25% </td><td> &gt; 25% </td></tr><tr><td> Postura </td><td> Normal </td><td> Hunching apenas em repouso </td><td> Hunching várias; prejudica movement </td></tr><tr><td> Atividade </td><td> Normal </td><td> Ligeira a moderada diminuição de actividade </td><td> Estacionário, a menos que estimulou </td></tr><tr><td> Textura da pele </td><td> Normal </td><td> Leve a moderada despenteando </td><td> Ruffling grave / preparação pobres </td></tr><tr><td> A textura da pele </td><td> Normal </td><td> Escala de patas / cauda </td><td> Áreas óbvias de pele desnudada </td></tr></tbody></table> Tabela 1. Cooke et al desenvolveram esse sistema de pontuação em 1996 1. Mice deve ser marcado diário em cada um dos critérios sobre a esquerda. Cada mouse é atribuída uma pontuação de 0-2 para cada critério ea pontuação total é a soma de todas as pontuações individuais. <img alt="Figura 1" src="/files/ftp_upload/3697/3697fig1.jpg" /> Figura 1. Letalmente irradiados foram transplantados BALB.B com 10 7 ossoAs células da medula sozinho ou com 18x10 6 células T CD8 esgotadas esplenócitos WT e 2x10 6 purificadas L2G85.B6 células T CD8. A) Os dados clínicos de pontuação receptores de medula óssea, isoladamente ou com células T CD8 esgotadas esplenócitos WT e purificado L2G85.B6 células T CD8. B) Os dados da sobrevivência de receptores de medula óssea sozinho ou com células T CD8 esgotado esplenócitos WT e purificado L2G85.B6 células T CD8. <a href="http://www.jove.com/files/ftp_upload/3697/3697fig1large.jpg" target="_blank">Clique aqui para ver maior figura</a> . <img alt="Figura 2" src="/files/ftp_upload/3697/3697fig2.jpg" /> Figura 2. Ratinhos BALB.B letalmente irradiados foram transplantados com 10 7 células de medula óssea por si só ou com 18x10 6 células T CD8 esgotadas esplenócitos WT e 2x10 6 purificadas L2G85.B6 células T CD8. Os receptores foram injectados com 4 mg de D-luciferina, por injecção intraperitoneal e foram fotografadas usando IVIS Xenogen durante 5 min a binning pequena. Pseudo cor-de-imagens são mostradas em roxo representa baixa intensidade e vermelha representa Regiões de alta intensidade de interesse foram desenhada ao redor do rato inteiro e fluxo total (fótons / s) foram quantificadas. <img alt="Figura 3" src="/files/ftp_upload/3697/3697fig3.jpg" /> Figura 3. Ratinhos BALB.B letalmente irradiados foram transplantados com 10 7 células de medula óssea por si só ou com 18x10 6 células T CD8 esgotadas esplenócitos WT e 2x10 6 purificadas L2G85.B6 células T CD8. Os receptores foram injectados com 4 mg de D-luciferina, por injecção intraperitoneal e foram fotografadas usando IVIS Xenogen durante 5 min a binning pequena. Pseudo-coloridas imagens são mostradas em roxo representa baixa intensidade e vermelha representa alta intensidade. Os destinatários foram fotografadas em um dia) 4, B) dia 6, e C) dias após transferência 8.

<ol>
	<li>Cooke, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+of+idiopathic+pneumonia+syndrome+after+bone+marrow+transplantation:+The+roles+of+minor+H+antigens+and+endotoxin.">An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: The roles of minor H antigens and endotoxin.</a> Blood. 88, 3230-3239 (1996).</li><li>Berger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+subsets+involved+in+lethal+graft-versus-host+disease+directed+to+immunodominant+minor+histocompatibility+antigens.">T cell subsets involved in lethal graft-versus-host disease directed to immunodominant minor histocompatibility antigens.</a> Transplantation. 57, 1095-1102 (1994).</li><li>Nimer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+depletion+of+CD8++cells+for+prevention+of+graft-versus-host+disease+after+bone+marrowtransplantation.+A+randomized+controlled+trial.">Selective depletion of CD8+ cells for prevention of graft-versus-host disease after bone marrowtransplantation. A randomized controlled trial.</a> Transplantation. 57, 82-87 (1994).</li><li>Korngold, R., Sprent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+markers+of+T+cells+causing+lethal+graft-vs-host+disease+to+class+I+vs+class+II+H-2+differences.">Surface markers of T cells causing lethal graft-vs-host disease to class I vs class II H-2 differences.</a> Journal of Immunology. 135, 3004-3010 (1985).</li><li>Cao, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+using+labeled+donor+tissues+reveals+patterns+of+engraftment,+rejection,+and+survival+in+transplantation.">Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation.</a> Transplantation. 80, 134-139 (2005).</li><li>Asady, R. E. l. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-{beta}-dependent+CD103+expression+by+CD8(+)+T+cells+promotes+selective+destruction+of+the+host+intestinal+epithelium+during+graft-versus-host+disease.">TGF-{beta}-dependent CD103 expression by CD8(+) T cells promotes selective destruction of the host intestinal epithelium during graft-versus-host disease.</a> J. Exp. Med. 201, 1647-1657 (2005).</li><li>Larson, R. S., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+leukocyte+integrins.">Structure and function of leukocyte integrins.</a> Immunol Rev. 114, 181-217 (1990).</li><li>Karecla, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+E-cadherin+on+epithelial+cells+by+the+mucosal+T+cell+integrin+alpha+M290+beta+7+(alpha+E+beta+7).">Recognition of E-cadherin on epithelial cells by the mucosal T cell integrin alpha M290 beta 7 (alpha E beta 7).</a> Eur. J. Immunol. 25, 852-856 (1995).</li><li>Cepek, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+between+epithelial+cells+and+T+lymphocytes+mediated+by+E-cadherin+and+the+alpha+E+beta+7+integrin.">Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the alpha E beta 7 integrin.</a> Nature. 372, 190-193 (1994).</li><li>Malarkannan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+and+functional+characterization+of+a+dominant+minor+H+antigen,+H60.">The molecular and functional characterization of a dominant minor H antigen, H60.</a> J. Immunol. 161, 3501-3509 (1998).</li></ol>

Não há conflitos de interesse declarados.

O protocolo para a indução de GVHD em ratinhos apresentados aqui representa um modelo clinicamente relevante da GVHD de murino. Originalmente criado por Berger et al., Em 1994, o C57Bl / 6 em BALB.B combinação estirpe é MHC-combinado, com mortalidade DECH mediada por CD4, CD8 dependentes efetores T 2, altamente semelhantes ao cenário clínico mais comum 3. Sabe-se que o transplante de células T CD8 sozinho não causar GVHD neste modelo, no entanto, a progressão da doença é signif...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 12633-012 </td><td></td></tr><tr><td> Fetal Calf Serum </td><td> Invitrogen </td><td> 10439016 </td><td></td></tr><tr><td> 40 Filtro celular mM </td><td> BD Biosciences </td><td> 352340 </td><td></td></tr><tr><td> CD3e-Biotina </td><td> Miltenyi Biotech </td><td> 130-093-021 </td><td></td></tr><tr><td> Anti-biotina de micropérolas </td><td> Miltenyi Biotech </td><td> 130-091-147 </td><td></td></tr><tr><td> CD8a micropérolas </td><td> Miltenyi Biotech </td><td> 130-049-401 </td><td> Usado para esgotar as células T CD8 de baço. </td></tr><tr><td> CD8a Cocktail Antibody Purification </td><td> Miltenyi Biotech </td><td> 130-095-236 </td><td> Usado para purificarCélulas T CD8 de baço. </td></tr><tr><td> D-luciferina </td><td> Caliper Life Sciences </td><td> 122796 </td><td></td></tr></tbody></table>

induction of graft-versus-host disease and in vivo t cell monitoring using an mhc-matched murine model

Enxerto-versus-hospedeiro (GVHD) é a barreira limitante à ampla utilização de transplante de medula óssea como uma terapia curativa para uma variedade de deficiências hematológicas. GVHD é causada por células T maduras alorreactivas presentes no enxerto de medula óssea que são infundidas no receptor e causar danos aos órgãos de acolhimento. No entanto, em ratinhos, as células T deve ser adicionado ao inoculo de medula óssea para causar GVHD. Apesar de extenso trabalho tem sido feito para caracterizar células T após o transplante respostas, bioluminescente tecnologia de imagem é um método não-invasivo para monitorar padrões de tráfico de células T in vivo. Após irradiação letal, ratinhos receptores foram transplantados com células de medula óssea e de esplenócitos de ratinhos dadores. Subconjuntos de células T a partir de L2G85.B6 (ratinhos transgénicos que expressam constitutivamente luciferase) estão incluídos no transplante. Por apenas transplantar certos subgrupos de células T, um é capaz de rastrear específicas subpopulações de células T in vivo,e com base na sua localização, desenvolver hipóteses sobre o papel das subpopulações de células T específicas para a promoção GVHD em vários pontos temporais. No transplante predeterminado intervalos post, ratos receptores são fotografada usando um Xenogen IVIS câmera CCD. A intensidade da luz pode ser quantificado usando software imagem viva para gerar uma imagem pseudo-cor com base na intensidade de fótons (vermelho = intensidade alta, violeta = intensidade baixa). Entre 4-7 dias após o transplante, os ratos receptores começam a apresentar sinais clínicos da GVHD. Cooke et al. 1 desenvolveu um sistema de pontuação para quantificar a progressão da doença com base na textura da pele destinatário ratos, a integridade da pele, atividade, perda de peso e postura. Os ratinhos são marcados diariamente, e sacrificados quando se tornam moribundo. Ratinhos receptores tornam-se geralmente moribundo 20-30 pós-transplante dias. Modelos murinos são ferramentas valiosas para o estudo da imunologia da GVHD. Seletivamente transplante especial de células T subconjuntos almínimos para identificação cuidadosa dos papéis de cada subconjunto joga. De forma não invasiva de rastreamento respostas de células T in vivo adiciona outra camada de valor para modelos murinos de GVHD.

Watch this Scientific Journal Video about Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model at JoVE.com

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

O transplante de medula óssea murina é uma técnica amplamente utilizada para estudar os mecanismos imunológicos que regem enxerto-versus-hospedeiro da doença em seres humanos. A capacidade de monitorar padrões de tráfico de células T<em&gt; In vivo</em&gt; Permite a análise detalhada do desenvolvimento e perpetuação da resposta das células T durante a reacção do enxerto contra o hospedeiro da doença.

Indução de enxerto-versus-hospedeiro de Doenças e<em&gt; Em Vivo</em&gt; Monitoramento celular T Usando um modelo murino de MHC de correspondência

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

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JoVE Journal

Immunology and Infection

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

15.6K visualizações.  The Ohio State University Medical Center. O transplante de medula óssea murina é uma técnica amplamente utilizada para estudar os mecanismos imunológicos que regem enxerto-versus-hospedeiro da doença em seres humanos. A capacidade de monitorar padrões de tráfico de células T In vivo Permite a análise detalhada do desenvolvimento e perpetuação da resposta das células T durante a reacção do enxerto contra o hospedeiro da doença.

Vídeo: Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Transdução retroviral de receptor de células T no rato células T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Imunologia e Infecção

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Um<em&gt; Ex vivo</em&gt; Modelo de um Ataque de T-Cell dirigiu-Oligodendrocyte em Agudos fatias de cérebro

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

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

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

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 ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Modelo Câmara fêmur Janela para<em&gt; In Vivo</em&gt; Acompanhamento de celular na Murino de Medula Óssea

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

A<em&gt; In Vitro</em&gt; Modelo da barreira sangue-cérebro que usa espectroscopia de impedância: Um foco na interação celular T Cell-endotelial

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Na Vivo Ensaio para detecção de células T antígeno-específicas função citolítica usando um modelo de vacinação

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Um protocolo de aspiração da bolha para estudar células T humanas Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Um modelo de cultura primária de células bolsas serosas Ex Vivo frango para estudar a patogênese de doenças infecciosas bolsas serosas vírus

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

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

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

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

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

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

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Determinação de subconjuntos de regulação de células T no timo murino, pancreático drenagem do linfonodo e baço usando citometria de fluxo

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...