Os autores gostariam de agradecer ao Dr. Boris Reizis do Departamento de Microbiologia e Imunologia da Universidade de Columbia para discussões úteis. Gostaríamos também de agradecer ao Dr. Margaret Bynoe, Dr. Rodman Getchell e Deequa Mahamed do Departamento de Microbiologia e Imunologia da Faculdade de Medicina Veterinária, da Universidade Cornell de assistência técnica com citometria de fluxo e Lu Huang para obter ajuda com edição de vídeo. Os autores também desejam agradecer aos membros do Duhamel e laboratórios Weiss para a sua revisão crítica do manuscrito e produção de vídeo, e Stephanie Yazinski do laboratório Weiss para reprodução e genotipagem p53 - / - ratos. Experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pela Cornell University Animal Care Institucional e Comitê de uso. Esta pesquisa foi suportada em parte por fundos fornecidos pelo Departamento de Agricultura dos EUA, Pesquisa do Estado de Cooperativa, Educação e Serviço de Extensão da Saúde Animal, e Programa de Pesquisa de Doenças (para GED e RSW) e subvenções NIH R03 HD058220 e R01CA108773 (para RSW). O vídeo foi produzido usando in-house instalações pelo pessoal dos laboratórios Duhamel &amp; Weiss.

1. Dissecação de Tumor <ol><li> Para dissecção do tumor, uma toalha de papel ou panos cirúrgicos descartáveis ​​é colocado na plataforma de dissecção e pulverizadas com etanol 70%. </li><li> Camundongos para este protocolo são rotineiramente submetidos à eutanásia com CO 2. Posicione o mouse sacrificados no teclado dissecção e spray de ambos os lados do animal com o etanol. O mouse é mantida no lugar sobre o bloco dissecção por meio de quatro ou mais alfinetes inseridos através das pernas e pulverizado completamente com álcool 70%. </li><li> A pele sobre a linha média é cortado a partir do púbis até a base da cabeça usando uma tesoura esterilizada, evitando-se os músculos subjacentes e penetrando nas cavidades do corpo. </li><li> Abrir a cavidade abdominal com um novo par de tesouras e pinças esterilizadas, tomando cuidado para não danificar os órgãos internos. </li><li> Abrir a cavidade torácica dissecando e desviando a caixa torácica para revelar o tumor, sem danificar os vasos sanguíneos. </li><li> Separar o tumor de outros órgãos, e de transferência de um pedaço de aproximadamente 40-50% do total do tumor (cerca de até 1,5 X 0,8 X 0,8 cm) em PBS frio mantidos em gelo. </li><li> A parte restante do tumor podem ser guardadas para outros ensaios e uma necropsia completa pode ser realizada neste momento. </li></ol> 2. Linfoma de células de cultura <ol><li> Pulverizar o tubo que contém o tumor dissecados com etanol 70% e transferir para um armário de biossegurança. </li><li> Adicionar 10 mL de meio de cultura para um 150 milímetros 2 placa de Petri estéril. </li><li> Transferir a peça de tumor na placa de Petri por sucção suave, com uma pipeta. </li><li> Dobre duas agulha estéril 18 voltada para o lado chanfrado para fora. </li><li> Dissociar o tumor usando a agulhas tortas. </li><li> Redemoinho do prato, bata suavemente e coloque em um ângulo para coletar a suspensão de célula para um lado, evitando grandes pedaços de tumor. </li><li> Aspirar a suspensão celular e transferir para um tubo cônico de 50 mL, evitando grandes pedaços de tumor. </li><li> Lave a placa com 10 mL de médio e transferência para o mesmo tubo, evitando grandes pedaços de tumor. </li><li> Misture a suspensão de células composto por linfócitos e células do estroma delicadamente e transferir 5 mL para um balão de cultura de tecidos T-25 e 10 mL em um T-75 frasco de cultura de tecidos. Agite suavemente os frascos. </li><li> Use a suspensão de células restantes para fazer 2, 4, 10100 e 1000 diluições em 2,5 mL de volume final (a cultura de células em densidades diferentes, utilizando vários tipos de embarcações cultura maximiza a probabilidade de estabelecer uma linha de células T). </li><li> Placa de 2,0 mL de cada diluição em poços individuais de uma placa de 6 poços. Agite ligeiramente a placa. </li><li> Incubar as células por 48 h em atmosfera umidificada CO 2 no ar. </li></ol> 3. Alimentando células de linfoma <ol><li> Após 48 h de incubação, adicionar 5 mL de meio para o balão de T-25 e 10 mL de meio para o balão de T-75 ao longo da parede com o mínimo de perturbação das células. </li><li> Adicionar 2 ml de meio em cada poço da placa de 6 poços ao longo da parede com o mínimo de perturbação das células. </li><li> Incubar as células para um adicional de 72 h. </li></ol> 4. Passaging Células Linfoma <ol><li> Passagem inicial <ol><li> No final do quinto dia de incubação, as células são várias passagens na proporção de 1:1, mantendo os frascos originais e placas (manutenção da densidade das células a uma concentração de cerca de 3x106 mL / durante as passagens de primeira promove a criação bem sucedida de um linhagem celular contínua). <ol class="lalpha"><li> Para o balão T-25, a transferência de 5 mL da suspensão celular para um balão novo contendo 5 mL de meio. Adicionar 5 ml de meio para o balão original. </li><li> Para o balão T-75, a transferência de 10 mL de suspensão celular para um balão novo contendo 10 mL de meio. Adicionar 10 mL de meio para o balão original. </li><li> Para a placa de 6 poços, a transferência de 2 mL da suspensão de células de cada poço em poços correspondente de um novo prato contendo 2 mL de meio em cada poço. Adicionar 2 ml de meio em cada poço dos poços original. </li></ol></li><li> Incubar todos os frascos e as placas por mais 72 h. Em nossa experiência mais linhas de células são estabelecidos a partir destes uma passagem culturas (a presença de agregados celulares frouxamente aderido a suspensão sobre as células do estroma anexado é uma indicação precoce de uma linhagem celular). </li><li> Para as culturas com menos ou nenhuma agregados celulares frouxamente aderente suspensão, alimentar os frascos ou placas com cuidado substituindo cerca de 50% do meio de cada 72 h Para evitar a remoção de células da cultura original, vasos cultura ficam em pé e em repouso por pelo menos 5 min para que o sedimento de células na parte inferior por gravidade, antes de ligeira aspiração do meio sobrenadante. </li></ol></li><li> Manutenção de linhas celulares estabelecidas <ol><li> Linhagens de células estabelecidas são várias passagens em frascos novos e placas. Para T-25 e T-75 frascos, adicionar 5 mL do original oususpensão uma passagem em 5 mL de meio. Para 6-bem pratos, adicionar 3 mL da suspensão de células original ou uma passagem em 7 mL de meio em um frasco T-25. Alimentar e manter o original ou uma passagem de cultura, substituindo o volume removido com meio de cultura fresco. </li><li> Passagem linhagens estáveis ​​de células em novos frascos T-25. As linhas de células têm um tempo de duplicação de 18-24 h. As culturas são rotineiramente mantidos a uma densidade de 1-3x106 mL / e várias passagens a cada 3 dias. Adicionar o volume necessário de passagem da cultura anterior para novo meio para fazer até 10 mL. Alimentar e manter a cultura original. </li><li> Depois de algumas passagens, as células não aderentes se auto sustentar sem a células aderentes do estroma e um número menor de células aderentes são transportados a cada passagem. Além deste ponto, as culturas podem ser mantidas em cultura de tecidos não-tratados T-25 frascos. </li></ol></li></ol> 5. Congelamento e Recuperação de linhagens de células linfoma <ol><li> As linhas celulares são congelados em preparados na hora RPMI 1640, contendo 20% FBS e DMSO 10%. </li><li> Células de uma cultura de idade em 48 h T-25 frasco (cerca de 1.5x10 6 células / mL) são centrifugadas a 1500 rpm por 5 min à temperatura ambiente. Após descartar o sobrenadante, as células são ressuspensas em 2 mL de meio de muito frio. Dois volumes 1,0 mL de suspensão celular de cada frasco são colocados em frascos de congelamento, imediatamente congeladas a -80 ° C, e transferidos para o nitrogênio líquido no dia seguinte. </li><li> Células são recuperados a partir de armazenamento de nitrogênio líquido por descongelamento rápido e ressuspender o conteúdo em 10 mL de meio de cultura fresco. Alternativamente, as células recém-descongelado são lavadas uma vez em meio de cultura morna para retirar o DMSO antes de ressuspender em 10 mL de meio de cultura fresco. As células são cultivadas por pelo menos 72 h antes da passagem subsequente. </li></ol> 6. Resultados representante Um Atm - / - células T-line desenvolvido utilizando este protocolo e designado DWJ-Atm-1 foi caracterizada por coloração com anti-CD3-FITC, anti-CD4-APC e anti-CD8-PE e análise de citometria de fluxo (Figura 1). Mais de 90% das células T-CD3 foram, CD4 e CD8 triple-positivo. <img src="/files/ftp_upload/2598/2598fig1.jpg" alt="Figura 1" /> Figura 1. Caracterização de uma linha de células T desenvolvido utilizando este protocolo. Os marcadores de superfície celular de um Atm estabelecida - / - linha de células designadas DWJ-Atm-1 foram caracterizados por coloração com anti-CD3-FITC, anti-CD4-APC, anti-CD8-PE e análise de citometria de fluxo. Resumidamente, 1X10 6 células frescas foram lavadas uma vez em PBS fria (1500 rpm / 5 min), e incubadas a 4 ° C por 10 min com 200 mL de coquetel de anticorpos (cada anticorpo na diluição de 1:200 em PBS com BSA 1%. As células podem ser examinados imediatamente, enquanto fresca ou fixo para análise posterior. células fixas são preparadas pela adição de formalina tamponada neutra a uma concentração de 4% (vol / vol) final e incubadas a 4 ° C por 30 min, colhidas por centrifugação (1500 rpm / 5 min) e ressuspendidas em 200 mL de PBS com BSA 1%. Um total de 1 X 10 4 células foram analisados ​​usando um BD FACS Calibur analisador e CellQuest software (BD Biosciences, San Jose, CA).

<ol>
	<li>Barlow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atm-deficient+mice:+a+paradigm+of+ataxia+telangiectasia.">Atm-deficient mice: a paradigm of ataxia telangiectasia.</a> Cell. 86, 159-171 (1996).</li><li>Elson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+defects+in+ataxia-telangiectasia+protein-deficient+mice.">Pleiotropic defects in ataxia-telangiectasia protein-deficient mice.</a> Proc Natl Acad Sci U S A. 93, 13084-13089 (1996).</li><li>Kuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Atm-/-+thymic+lymphoma+cell+proliferation+in+vitro+and+in+vivo+by+dexamethasone.">Control of Atm-/- thymic lymphoma cell proliferation in vitro and in vivo by dexamethasone.</a> Cancer Chemother Pharmacol. 55, 203-212 (2005).</li><li>Browne, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+a+catalytic+antioxidant+corrects+the+neurobehavioral+defect+in+ataxia-telangiectasia+mice.">Treatment with a catalytic antioxidant corrects the neurobehavioral defect in ataxia-telangiectasia mice.</a> Free Radic Biol Med. 36, 938-942 (2004).</li><li>Jacks, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+spectrum+analysis+in+p53-mutant+mice.">Tumor spectrum analysis in p53-mutant mice.</a> Curr Biol. 4, 1-7 (1994).</li><li>Chervinsky, D. S., Lam, D. H., Zhao, X. F., Melman, M. P., Aplan, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+T+cell+leukemia+cell+lines+established+from+SCL/LMO1+double+transgenic+mice.">Development and characterization of T cell leukemia cell lines established from SCL/LMO1 double transgenic mice.</a> Leukemia. 15, 141-147 (2001).</li><li>Sharma, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1+contributes+to+mouse+T-cell+leukemia+by+directly+inducing+the+expression+of+c-myc.">Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc.</a> Mol Cell Biol. 26, 8022-8031 (2006).</li><li>Maser, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomally+unstable+mouse+tumours+have+genomic+alterations+similar+to+diverse+human+cancers.">Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers.</a> Nature. 447, 966-971 (2007).</li></ol>

Não há conflitos de interesse declarados.

Neste protocolo, nós fornecemos os procedimentos detalhados para estabelecer murino de células T linhas de dois genótipos diferentes do mouse; Atm - / - [FVB/N- Atm tm1Led / J] p53 e - / - [129/S6- Trp53 tm1Tyj / J] . Usando esse protocolo, um total de 6 (de 7 tentativas) Atm - / - e três (de 5 tentativas) p53 - / - murino de células T linhas foram estabelecidas em nosso laboratório. Dad...

<ul><li> Balde de gelo </li><li> Etanol 70% </li><li> Pad dissecção consistindo de bloco de espuma coberto com papel alumínio e pinos </li><li> Dois conjuntos de pequenas tesouras e pinças esterilizadas, </li><li> Estéril 150 milímetros 2 Placa de Petri (Thermo Fisher Scientific, Waltham, MA;. Cat # 08-757-13) </li><li> 18 agulhas estéreis </li><li> 50 mL tubos cônicos (Corning Inc., Corning NY; cat # 430829) </li><li> 10% de formalina tamponada </li><li> Cultura de tecidos frascos <ul><li> T-25 (TPP, Trasadingen, Suíça; cat # 90025) </li><li> T-75 (TPP, Trasadingen, Suíça; cat # 90075) </li></ul></li><li> 6-lamelas (Corning Inc., Corning NY; cat # 3506) </li><li> Fosfato estéril tamponada pH 7,2 solução salina (PBS) </li><li> Meio de cultura <ul><li> RPMI 1640 com 25 HEPES mM, 200 mM L-glutamina (Lonza, Walkersville, MD; gato # 12-115F), suplementado com o seguinte: <ul><li> Calor de 10% inativado (56 ° C, 30 min) de soro fetal bovino (FBS; Hyclone, Logan, UT; # cat SH30088.03) </li><li> 1% de penicilina e estreptomicina (Mediatech, Manassas, VA; gato # 30-002-CI) </li><li> 1% aminoácidos não essenciais (Mediatech; gato # 25-025-CI) </li><li> 55 mM 2β-mercaptoetanol (Sigma, St Louis, MO; cat # M752) </li></ul></li></ul></li><li> Dimetil sulfóxido (DMSO; Calbiochem, La Jolla, CA; cat # 317275) </li><li> Anticorpos (eBioscience, San Diego, CA) <ul><li> anti-CD3-FITC (cat. # 11-0031-85) </li><li> anti-CD4-APC (cat. # 17-0041-83) </li><li> anti-CD8-PE (cat. # 12-081-85) </li></ul></li><li> Albumina sérica bovina (BSA; Life Technologies, Grand Island, NY;. Cat # 11018-017) </li><li> Concanavalina A (Sigma cat # C5275) </li><li> 1,8 mL frascos de congelamento (Nunc, Roskilde, na Dinamarca; cat # 368632) </li></ul>

derivation of thymic lymphoma t-cell lines from atm-/- and p53-/- mice

As linhas celulares são uma ferramenta de pesquisa fundamental que pode reduzir o uso de animais de laboratório em pesquisa. Certas cepas de camundongos geneticamente modificados, tais como ATM - / - e p53 - / - consistentemente desenvolver linfoma tímico no início da vida 1,2, e, portanto, pode servir como uma fonte confiável para a derivação de células T murino linhas. Aqui apresentamos um protocolo detalhado para o desenvolvimento do timo murino estabelecida linfoma de células T linhas sem a necessidade de adicionar interleucinas, conforme descrito nos protocolos anteriores 1,3. Tumores foram colhidas de camundongos com idade entre três a seis meses, na primeira indicação de tumores visíveis a partir da observação da postura arqueada, trabalhou preparação respiração, pobres e desperdício em um 1,4 linhagem suscetível. Nós temos estabelecido com sucesso várias linhas de células T usando este protocolo e de estirpes puras e Atm - / - [FVB/N- Atm tm1Led / J] 2 e p53 - / - [129/S6- Trp53 tm1Tyj / J] 5 camundongos. Nós ainda demonstram que mais de 90% do estabelecido população de células T CD3 expressa, CD4 e CD8. Consistente com linhas de células de maneira estável estabelecida, as células-T gerados usando o protocolo presentes foram várias passagens por mais de um ano.

Watch this Scientific Journal Video about Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice at JoVE.com

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Neste vídeo mostramos um protocolo para estabelecer linhagens de células do timo de rato linfoma. Ao seguir este protocolo, conseguimos estabelecer várias células T linhas de Atm-/ - e p53-/ - ratos com linfoma tímico.

Derivação de Tímico linfoma de células T Linhas de Atm - / -</sup E<emP53> - / -</sup Ratos

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

derivation-of-thymic-lymphoma-t-cell-lines-from-atm-and-p53-mice

Research

JoVE Journal

Immunology and Infection

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

12.2K visualizações.  Cornell University. Neste vídeo mostramos um protocolo para estabelecer linhagens de células do timo de rato linfoma. Ao seguir este protocolo, conseguimos estabelecer várias células T linhas de Atm-/ - e p53-/ - ratos com linfoma tímico.

Vídeo: Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Ratos Transnuclear com pré-definidos T Especificidades receptor da célula contra a Toxoplasma gondii Obtidos Via TNCS

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Imunologia e Infecção

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 ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

Exame de seleção positiva e negativa Tímico por citometria de fluxo

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Em tempo real de imagens ao vivo de células T Sinalização de Formação de Complexos

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

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 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Derivação de células T<em&gt; In Vitro</em&gt; De Mouse Células-Tronco Embrionárias

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

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

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

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

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

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

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

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Transdução retroviral de Células de Medula Óssea progenitoras para gerar células T Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

Preparação e Aplicações de culturas organotípicas Tímico Fatia

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

Um método simples e eficiente para estabelecer linhas de células T de pacientes com infecção crônica ativa vírus Epstein - Barr e NK

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Derivação de Tímico linfoma de células T Linhas de Atm - / - E - / - Ratos

Derivação de Tímico linfoma de células T Linhas de Atm^{- / - E^{- / - Ratos}}