EJV é apoiado por uma bolsa da s &amp; Colite de Crohn Foundation of America (CCFA). JRM é suportado por concessões do CCFA, Cancer Research Institute (CRI), H. Howard Goodman (MGH), Life Science Center Massachusetts (MLSC) e Prêmio NIH Director s New Innovator.

1. Geração ex vivo de células T gut-homing e controle (ver Figura 1) <ol><li> Isolar esplenócitos por mashing um baço de camundongos de tipo selvagem. Volte a suspender as células em suspensão PBS e centrifugar por 5 &#39;a 400 x g. Remover o sobrenadante e lise das células vermelhas do sangue pela ressuspensão do pellet celular em 4 ml de tampão de lise ACK por 2-3 minutos. Depois disso, adicione 5 ml de PBS. Centrifugar por 5 &#39;a 400 xg e remover o sobrenadante. </li><li> Ressuspender esplenócitos total em 1 x 10 6 / mL em IMDM + 10% FBS + 50 mM 2-mercaptoetanol + penicilina / estreptomicina (IMDM completa). Separar as células em dois grupos, um dos quais são complementados com RA (ou sintético RAR-agonistas, tais como Am80 ou Am580) para uma concentração final de 100-200 nM 3. Células T ativadas na presença de RA ou RAR-agonistas vai upregulate a4b7and CCR9 e vai se tornar células do intestino trópico-T, enquanto que as células T ativadas sem RA vai se tornar as células T de controle. </li><li> Adicionar 1,5-2,0 mL da suspensão de células em cada poço de uma placa de 24 poços previamente revestidas com anti-CD3 (10 mg / ml) e anti-CD28 (10 mg / ml) e incubar a 37 ° C em 5% de CO 2 (para o revestimento da placa de adicionar 500 ml / poço de anti-CD3-CD28, mais anti em PBS e incubar por 2 horas a 37 ˚ C e depois lave duas vezes com 2 ml PBS). </li><li> A fim de evitar a superestimulação das células T, após 2-3 dias de transferência das suspensões de células em um novo sem revestimento placa de 24 cavidades. Incubar por mais de 2-3 dias. Dependendo da densidade e proliferação celular a mídia pode tornar-se ácido (amarelo), caso em que pode ser necessário para substituir a metade da mídia com IMDM completa fresco. Colheita das células após 4-5 dias (contando do dia 0). </li><li> Típico rendimentos finais são 1-2 x 10 6 células T efectoras / poço. Portanto, um mínimo de 12/09 poços de cada células do intestino trópico-T e células T de controle deve ser banhado, a fim de acabar com 10-20 x 10 6 células T por condição. </li></ol> 2. Análise de gut-homing receptores nas células T ativadas Após 4-5 dias de cultura, devemos observar uma alta expressão de receptores gut-homing a4b7and CCR9 em células T ativadas na presença de agonistas RA ou RAR, enquanto que as células T de controle deve expressar níveis muito baixos destas quatro receptores. Citometria de fluxo análise (FACS). <ol><li> Coletar entre 0,2-0,5 x10 6 células e centrifugar por 5 min a 300 g a 4 ° C. </li><li> Incube com anti-CD4-FITC, anti-a4b7-PE (BD Biosciences), anti-CCR9-APC (eBiosciences) e anti-CD8-PerCP por 15 min a 4 ° C no escuro. </li><li> Após a incubação, centrifugar por 5 min a 300 g 4 ° C. Lavar e ressuspender as células em tampão de coloração e análise por FACS. </li></ol> 3. T rotulagem celular com células trackers CFSE e CMTMR <ol><li> Manter todas as soluções a 37 ° C antes de começar. Lavar as células do intestino-trópico T (a4b7 alta CCR9 Alta) e células controle T (a4b7 Baixo / Neg CCR9 Baixo / Neg) duas vezes com PBS para remover o soro. Ajuste a concentração de células T para 10-15 x 10 6 / mL em PBS. </li><li> Adicionar CFSE (carboxifluoresceína succinimidyl éster) ou CMTMR (clorometil-benzoil-amino-tetrametilrodamina) fluoróforos para qualquer gut-trópico T e células T de controle para uma concentração final de 5 mm e 10 mm, respectivamente, gentilmente vortex e incubar por 20 min em 37 ° C. Recomenda-se trocar o bulas no mesmo ou em um experimento separado, a fim de excluir potenciais efeitos intrínsecos de fluoróforos em T migração celular. </li><li> Após a incubação, saciar com um volume de FBS e incubar por 1-5 min à temperatura ambiente. Diluir 10 vezes com PBS quente e centrifugar por 5 min a 300 g. Lave duas vezes com PBS e ressuspender em IMDM completa. </li><li> Idealmente, 10-20 x 10 6 células de cada população T deve ser misturado na proporção de 1:1 e, em seguida, centrifugado a 300 xg por 5 min. Finalmente, voltar a suspender as células em 200-250 mL de PBS quente e injetar através da veia da cauda. </li><li> Para calcular a taxa de entrada exata (de preferência perto de 1) salvar 5-10 ml da suspensão de células injetadas e diluir com 300 mL de PBS e analisar por FACS. </li></ol> 4. Análise de células T homing Embora os ratos podem ser analisados ​​em vários pontos do tempo (entre 1 h e vários dias após a injeção de células T), homing das células do intestino trópico-T é o melhor documentado entre 12-24 h após a injecção. Pontos mais tempo, aumentar os potenciais efeitos de outras variáveis ​​que afetam a célula final números / índices, como a apoptose de células T e saída de células T do tecido. Depois de os ratos são sacrificados, suspensões de células de vários tecidos devem ser isolados, sem demora, a fim de diminuir a morte celular, que pode afetar o rendimento final e resultados. Tecidos de interesse são o intestino delgado, cólon, baço, linfonodos, placas de Peyer, fígado, pulmões e sangue. Se oinjetaram células T são bona fide células do intestino trópico-T, devem casa, em média 5-10 vezes melhor (ou superior) para a mucosa do intestino delgado em relação ao controle de células T. No entanto, outros tecidos, como sangue e do baço, não deve apresentar uma migração de células T preferencial. Isolamento de linfócitos da lâmina própria intestinal eo compartimento intra-epitelial tem sido descrito anteriormente 5. <ol><li> Coloração FACS: As amostras são ressuspenso dependendo do número de células coletadas, com uma densidade não superior a 3,0 x 10 6 células / ml. Se congênitas CD45.1 + ou + Thy1.1 ratos foram usados ​​como receptores, as células são corados com o marcador correspondente congênitas (por exemplo, ou CD45.2 Thy1.2) combinado com algum marcador de linhagem (por exemplo, TCRbchain, CD4, CD8 , CD45.1 + / CD45.2 +). </li><li> Durante a análise do FACS, as células são fechadas no marcador congênitas (por exemplo, CD45.1) e, em seguida, os marcadores de linhagem específica (por exemplo, CD4/CD8) e analisadas para as relações entre CMTMR e células CFSE positivo. </li><li> Os dados é geralmente expressa como o Índice de Homing (HI), que é calculado como a relação CFSE / CMTMR (ou CMTMR / CFSE) em cada tecido dividido pela relação de entrada correspondente (veja abaixo). Se a relação de entrada é muito próximo de 1, então as razões de tecido será equivalente à HI. </li></ol> HI = CFSE tecido tecido / CMTMR: Entrada CFSE input / CMTMR De sangue também devem ser analisados ​​a fim de determinar se as duas populações de células T são igualmente representados na circulação ou se uma população de células T está diminuído relação ao outro (por exemplo, prendendo preferencial nos pulmões / fígado ou por viabilidade diminuída). Isso pode acontecer quando se comparam as células de memória T naïve ou descansando contra recentemente activado células T efetoras, já que este último pode ser preso em maior medida nos pulmões. Se o HI no sangue é significativamente diferente de 1, pode-se normalizar HI nos tecidos pelo HI no sangue. Sangue pode ser obtido através de punção cardíaca de ratos anestesiados (com Avertin ou isofluorano eutanásia antes) e devem ser lisadas por duas vezes com tampão ACK antes de o utilizar para a coloração FACS. Normalização de sangue = HI Sangue tecido / HI 5. Resultados representante Camundongos destinatário (Thy1.1 +) foram euthanized18h injeção de células post. Suspensões de células foram gerados a partir do baço, linfonodos periféricos (PLN), linfonodos mesentéricos (MLN) e lâmina própria do intestino delgado (LP). Depois que as células foram coradas para Thy1.2 e CD8 e depois analisados ​​por FACS pela passagem de células viáveis ​​Thy1.2 + CD8 +, que finalmente foram analisadas para a relação entre as células CMTMR + (células T gut-trópico) e CFSE + (células de controlo T) dividido pela relação de entrada. Os resultados podem ser exibidos como mostrado na Figura 2, em que pode ser apreciado que as células do intestino trópico e controle T homed igualmente para o baço (HI perto para 1, indicada por uma linha vermelha). Em contraste, as células do intestino trópico-T migraram cerca de 10 vezes mais eficiente para o LP em relação ao controle de células T. <img src="/files/ftp_upload/2619/2619fig1.jpg" alt="Figura 1" /> Figura 1:. Diagrama mostrando a geração e rotulagem de gut-tropical e não-gut células T trópico células T são isolados de camundongos do tipo selvagem e activado com anti-CD3/anti-CD28 e na presença ou ausência de 100-200 nM todos - trans ácido retinóico (AR). Depois de 4-5 dias RA-tratados células T adquirir a expressão de receptores do intestino homing CCR9 e a4b7. Então, as células do intestino trópico e controle T são diferencialmente marcada com CFSE ou CMTMR, lavado, misturado na proporção de 1:1 e injetado em um rato destinatário através da veia da cauda. Algumas células marcadas são usadas para determinar a relação de entrada CFSE / CMTMR (deve ser próximo a 1). <img src="/files/ftp_upload/2619/2619fig2.jpg" alt="Figura 2" /> Figura 2:. Mice Exemplo de análise homing analisados ​​12-18 h pós-injeção de células T e suspensões única célula são obtidos a partir dos órgãos de interesse. As células são corados com anti-Thy1.2 (marcador congênitas) mais anti-CD8 + e depois Thy1.2 células T CD8 + são analisados ​​para a relação entre CMTMR + e + CFSE células em cada tecido por FACS. O tecido CMTMR / CFSE índices são normalizados pela entrada relação CMTMR / CFSE para obter os índices de homing (HI). Neste + CMTMR exemplo e CFSE células são + gut-tropical e as células T efetoras controle, respectivamente.

<ol>
	<li>Butcher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-endothelial+cell+recognition:+Three+(or+more)+steps+to+specificity+and+diversity.">Leukocyte-endothelial cell recognition: Three (or more) steps to specificity and diversity.</a> Cell. 67, 1033-1036 (1991).</li><li>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=Traffic+signals+for+lymphocyte+recirculation+and+leukocyte+emigration:+the+multistep+paradigm.">Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.</a> Cell. 76, 301-314 (1994).</li><li>Iwata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+imprints+gut-homing+specificity+on+T+cells.">Retinoic acid imprints gut-homing specificity on T cells.</a> Immunity. 21, 527-538 (2004).</li><li>Mora, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+and+dynamic+control+of+CD8+T+cell+homing+by+dendritic+cells+from+skin-+and+gut-associated+lymphoid+tissues.">Reciprocal and dynamic control of CD8 T cell homing by dendritic cells from skin- and gut-associated lymphoid tissues.</a> J Exp Med. 201, 303-316 (2005).</li><li>Mora, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imprinting+of+gut-homing+T+cells+by+Peyer's+patch+dendritic+cells.">Selective imprinting of gut-homing T cells by Peyer's patch dendritic cells.</a> Nature. 424, 88-93 (2003).</li><li>Mempel, T. R., Scimone, M. L., Mora, J. R., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+leukocyte+trafficking+in+blood+vessels+and+tissues.">In vivo imaging of leukocyte trafficking in blood vessels and tissues.</a> Curr Opin Immunol. 16, 406-417 (2004).</li></ol>

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Apesar de experimentos homing fornecer informações valiosas sobre a migração de populações de células totais em qualquer dado tecido, ele deve ser mantido em mente que estes ensaios não analisam diretamente adesão endotelial e, portanto, não discriminar em qual etapa (s) da adesão de várias etapas cascata (tethering / rolamento, ativação ou furar) um determinado receptor homing está agindo. O padrão ouro para definir o papel específico dos receptores de homing na cascata de adesão é de microscopia int...

Animais: camundongos C57BL / 6 são comumente usados ​​para o isolamento de células T. Além disso, CD45.1 e Thy1.1 linhagens congênitas estão disponíveis através de Jackson Laboratories (Bar Harbor, ME). Cultura da mídia: IMDM (Medium Modificado Iscove de Dulbecco + L-Glutamina + Hepes) acrescido de 10% inativado pelo calor FBS (fetal bovino endotoxina, Serum baixa, Gibco ®, Invitrogen, Carlsbad, CA) suplementado com 100 U / penicilina ml, 100 mg / ml de estreptomicina (antibióticos HyClone, Waltham, MA), 0,5 mg / ml Fungizone / anfotericina B (Gibco) e 50 mM b-mercaptoetanol. ACK Red Blood tampão de lise celular (RBC, 10 mM KHCO 3, 150 mM NH 4 Cl, 0,1 mM EDTA, pH 8,0), ajustado a pH 7,2-7,4 e armazenar em temperatura ambiente). PBS (Phosphate Buffered Saline, Hyclone, Waltham, MA). Citometria de fluxo (FACS) media (PBS ou IMDM + 2 + 5% FBS mM EDTA). Quando coloração com Selectina-Fc quimeras, media com 2 mM Ca + + deve ser usado em todas as etapas (incluindo a aquisição FACS). IMDM é recomendado neste caso. T rotulagem celular e transferência adotiva: CFSE (carboxifluoresceína diacetato, succinimidyl éster), CMTMR ((5 - (e-6 )-((( 4-clorometil) benzoil) amino) tetrametilrodamina) de Molecular Probes ®, Invitrogen, Carlsbad, CA ). 1.000 ações x deve ser feita em DMSO (5 CFSE mM, 20 mM CMTMR) e armazenadas a -20 ° C. Ativação de células T policlonais: 24 bem ou placas de 96 poços (cultura de tecidos tratados, poliestireno, de fundo plano com tampa, BD Falcon, Franklin Lakes, NJ) são incubados por 2 horas a 37 ° C com 50 ml de PBS, respectivamente , contendo anti-CD3, mais anti-CD28 anticorpos (10 mg / mL cada). Então, placas de cultura são lavados duas vezes com PBS e imediatamente utilizado para cultura de células T. Alternativamente, Dynabeads revestido com anti-CD3/anti-CD28 (Dynal, Invitrogen, Carlsbad, CA) pode ser usada para ativação das células T, em vez de placa-bound anticorpos. Citometria de fluxo coloração (FACS): ativação policlonal: CD3 (1452C11), CD28 (37,51). Lineage mAb: CD4 (L3T4), CD8a (Ly-2), Thy1.2 (CD90.2/53-1.2), CD45.2 (104). Gut-homing receptores: purificada CCR9 (CD199/eBioCW-1.2, eBioscience, San Diego, CA), a4b7 (LPAM-1/DATK3), isotipo controle (IgG2a, k). Pele-homing receptores: P-selectina-Fc (Purified rato P-Selectina - IgG proteína de fusão, BD PharMingen, San Jose, CA), E-selectina-Fc (E-Selectin/Fc rato recombinante Chimera, R &amp; D Systems, Minneapolis, MN), mais de cabra F correspondente secundário reagente (ab &#39;) 2 anti-humano IgG R-PE (Invitrogen, Carlsbad, CA). All-trans ácido retinóico (Sigma, St. Louis, MO) é ressuspenso em etanol absoluto ou DMSO usando uma lâmpada amarela ou uma fonte indireta de luz durante a preparação. Alíquotas armazenar em frascos de vidro a -80 ° C e protegida da luz em todos os momentos. Sintéticos RAR-agonistas: Am80 (Wako Chemicals, Richmond, VA).

competitive homing assays to study gut-tropic t cell migration

A fim de exercer sua função linfócitos necessidade de deixar o sangue e migram para diferentes tecidos no organismo. Adesão dos linfócitos às células endoteliais e extravasamento de tecido é um processo de várias etapas controladas por diferentes moléculas de adesão (receptores homing) expressa em linfócitos e seus ligantes respectivos (addressins) exibidos em células endoteliais 1 2. Mesmo que a função desses receptores de adesão pode ser parcialmente estudou ex vivo, o teste final para sua relevância fisiológica é avaliar o seu papel durante na adesão de linfócitos vivo e migração. Duas estratégias complementares têm sido utilizados para esse fim: microscopia intravital (IVM) e experimentos homing. Embora IVM tem sido essencial para definir a contribuição precisa de receptores de adesão específicos durante a cascata de adesão em tempo real e em diferentes tecidos, IVM é demorado e trabalhoso, que muitas vezes requer o desenvolvimento de sofisticadas técnicas cirúrgicas, que necessita de isolamento antes de homogêneo populações de células e permite a análise de apenas um órgão, tecido / a qualquer momento. Pelo contrário, competitiva experimentos homing permitir a comparação direta e simultânea na migração de duas (ou mais) em subpopulações de células com o mouse mesmo e eles também permitem a análise de muitos tecidos e de um elevado número de células no mesmo experimento. Aqui nós descrevemos o protocolo competitivo clássica homing usado para determinar a vantagem / desvantagem de um tipo de célula dado a casa para tecidos específicos, em comparação com uma população de células controle. Escolhemos para ilustrar as propriedades migratórias do intestino trópico-versus células não gut trópico-T, porque a mucosa intestinal é a superfície maior corpo em contato com o ambiente externo e também é o tecido extra-linfóides com os requisitos definidos pelo melhor migratórias . Além disso, trabalhos recentes determinou que a vitamina A, ácido all-trans retinóico metabólito (RA) é o principal mecanismo molecular responsável pela indução de receptores específicos gut-adesão (integrinas receptor de quimiocina a4b7and CCR9) em linfócitos. Assim, podemos facilmente gerar um grande número de gut-tropical e não gut trópico de linfócitos ex vivo pela ativação de células T na presença ou ausência de RA, respectivamente, o que pode ser finalmente utilizado nos experimentos competitivos homing descrito aqui.

Watch this Scientific Journal Video about Competitive Homing Assays to Study Gut-tropic T Cell Migration at JoVE.com

Competitive Homing Assays to Study Gut-tropic T Cell Migration

Competitiva homing experimentos permitem avaliar diretamente as propriedades migratória de duas populações celulares diferentes em um único rato. Aqui nós ilustrar este procedimento através da comparação da migração de ex-vivo gerado gut trópico-versus non-gut células T trópico.

Competitiva Ensaios Homing para Estudar Gut trópico-Migração de Células T

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

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JoVE Journal

Immunology and Infection

12.4K visualizações.  Massachusetts General Hospital, Harvard Medical School. Competitiva homing experimentos permitem avaliar diretamente as propriedades migratória de duas populações celulares diferentes em um único rato. Aqui nós ilustrar este procedimento através da comparação da migração de ex-vivo gerado gut trópico-versus non-gut células T trópico.

Vídeo: Competitive Homing Assays to Study Gut-tropic T Cell Migration

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Isolamento T de linfócitos humanos, Cultura e Análise da Migração In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Imunologia e Infecção

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Não-invasiva por imagem do Homing leucócitos e Migração In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

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 ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Uma avaliação quantitativa da migração celular pelo ensaio Phagokinetic faixa Motilidade

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Transdução de adenovírus Naive CD4 células T para estudar a diferenciação Treg

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

O transplante de pele da cauda para estudar as respostas das células T CD4 alogênico em camundongos

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

Migração intersticial imagens de células CD4 T na derme inflamadas

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Um ensaio baseado em CFSE para estudar a migração de Murino pele as células dendríticas em drenagem gânglios linfáticos durante a infecção com<em&gt; Mycobacterium bovis</em&gt; Bacilo de Calmette-Guérin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

Usando cristalografia de raios X, Biofísica, e os ensaios funcionais para determinar a célula T mecanismos que regem o reconhecimento do receptor de Câncer Antígenos

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

Um romance em Vitro ferida cura ensaio para avaliar a migração celular

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Competitiva Ensaios Homing para Estudar Gut trópico-Migração de Células T

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