Os autores gostariam de agradecer a André Han e Yen Lawrence para assistência técnica, e os membros do laboratório Jenkins ajuda para o desenvolvimento deste protocolo.

1. Isolamento de células a partir de tecido linfóide <ol><li> Adicionar 1 ml de gelo frio cEHAA (EHAA + 10% FBS, pen / strep, gentamicina, 2 mM de L-glutamina, 55 mM 2-mercaptoetanol), ou outro meio equivalente de células T, para uma placa de cultura de 60 milímetros, contendo um pequeno quadrado de 100 malha de nylon fim e colocar no gelo. </li><li> Euthanize mouse. </li><li> Remover o baço e os linfonodos como muitos facilmente acessível quanto possível. Estes devem incluir pelo menos os inguinais, axilares, braquiais, linfonodos cervicais, e mesentérica. Colocá-las em cima da rede de nylon na placa de cultura. </li><li> Utilizando o plano superior de um sistema fechado de tubo de microcentrífuga de 1,5 ml, suavemente esmagar o tecido linfóide sobre a malha de nylon para libertar os linfócitos. Adicionar 1 ml de uma outra cEHAA e pipeta cima e para baixo para o trabalho de células numa suspensão. Transferir as células através de um outro pedaço de malha de nylon colocada sobre a parte superior de um tubo de centrífuga de 15 ml de polipropileno. Lave o prato e malha com outro 1 ml de gelo frio cEHAA, pooling os volumes no mesmo tubo. Repetir para atingir um volume de suspensão final de células de 4 ml. </li><li> Adicionar tampão classificador frio (PBS + Azida de 2% de FBS, 0,05%) a um volume final de 15 ml e centrifugar o tubo durante 5 minutos a 300 xg, 4 ° C. </li><li> Aspirar o sobrenadante cuidadosamente, certificando-se gotículas de líquido não são deixados sobre os lados do tubo. Ressuspender o sedimento de células em Fc bloco (classificador de tampão + 2.4G2 anticorpo) a um volume final igual a cerca de duas vezes maior que o pellet em si. Por exemplo, os gânglios linfáticos e do baço de um ratinho ingénuo geralmente produz um pelete de células de cerca de 100 uL. Nesse caso, adicionar 100 ul de bloco de Fc para levar o volume para 200 ul. Se um grande grau de aglomeração de células ocorreu, remova cuidadosamente o aglomerado de células neste momento com uma pipeta. </li></ol> 2. Coloração tetrâmero <ol><li> Adicionar PE-ou APC-rotulado pMHC tetrâmero para uma concentração final de 10 nM (concentração ou empiricamente optimizados). </li><li> Mix e incubaçãote durante 1 hora à temperatura ambiente (ou a hora empiricamente optimizados e temperatura). </li><li> Adicionar tampão classificador fria até um volume de 15 ml e centrifugar o tubo durante 5 minutos a 300 xg, 4 ° C. Manter as amostras em gelo ou a 4 ° C a partir de agora. </li><li> Aspirar o sobrenadante cuidadosamente, certificando-se gotículas de líquido não são deixados sobre os lados do tubo. Ressuspender o sedimento de células em tampão de classificador para um volume final de 200 ul. Para o enriquecimento de tetrâmero de casal, ressuspender a um volume final de 150 ul. </li></ol> 3. Enriquecimento magnética <ol><li> Adicionar 50 ul de microesferas de Miltenyi anti-PE ou anti-APC. Para o enriquecimento de tetrâmero de casal, adicionar 50 ul de ambas. </li><li> Misturar e incubar durante 20 min a 4 ° C. </li><li> Adicionar tampão classificador fria até um volume de 15 ml e centrifugar o tubo durante 5 minutos a 300 xg, 4 ° C. </li><li> Enquanto espera, configurar uma Miltenyi coluna LS magnético em um ímã MidiMACS ou QuadroMACS. Uma posição aberta 15 ml tubo de centrífuga de polipropileno com umacumular diretamente abaixo da coluna. </li><li> Adicionar 3 ml de tampão de classificador para a parte superior da coluna, o que permite que seja drenado para o tubo de 15 ml. </li><li> Coloque um quadrado de 100 um de malha de nylon no topo da coluna. </li><li> Quando as células de ter terminado a fiação, aspirar cuidadosamente o sobrenadante e ressuspender o sedimento em 3 ml de tampão de classificador. </li><li> Transferir a suspensão de células através da malha de nylon para o topo da coluna. </li><li> Quando a suspensão de células foi completamente drenada para a coluna, lavar o tubo inicial com um outro de 3 ml de tampão e transferir o classificador de tampão através da malha de nylon para a coluna, lavagem da malha durante o processo. Descarte a malha de nylon. </li><li> Quando o tampão foi completamente drenada para a coluna, adicionar 3 ml de um outro tampão classificador para a coluna. </li><li> Repetir o passo 10 para um total de 3 x 3 ml lavagens. </li><li> Remover a coluna do magnete e local ao longo de um novo tubo de 15 ml de centrífuga de polipropileno. </li><li> Adicionam-se 5 ml de tampão de classificador de tele coluna. </li><li> Imediatamente inserir o êmbolo no topo da coluna e com um movimento contínuo, empurre o êmbolo para baixo, forçando o tampão para fora da coluna dentro do tubo. </li><li> Centrifugar os tubos contendo a fracção eluída ligado e a fracção não ligada por escoamento durante 5 minutos a 300 xg, 4 ° C. </li><li> Aspirar cuidadosamente o sobrenadante a partir da fracção ligada, certificando-se gotículas de líquido não são deixados sobre os lados do tubo. Ressuspender o sedimento de células em tampão de classificador para um volume final de 95 ul exactamente. Aspirar e ressuspender a fracção não ligada a um volume final de 2 ml. </li></ol> 4. Citometria de Fluxo <ol><li> Para cada fracção, remover e adicionar 5 ul a 200 ul de pérolas de contagem (ajustada para uma concentração de 200,000 / ml em tampão sorter) em um tubo de 5 ml de FACS. Separe a 4 ° C para análise posterior. Para poupar tempo, os grânulos podem ser pré-aliquotadas para tubos marcados antes do início da experiência. </li><li> Prepare amaster mistura de anticorpos para marcadores de superfície de manchar as células (Tabela 1). Para poupar tempo, isto pode ser feito antes do início da experiência. </li><li> Para a fracção ligada, adicionar uma dose de cocktail de anticorpos directamente para o ~ 90 ul de células no tubo. Se a análise da fracção não ligada é desejada, transferir 90 ul de células para um tubo de 5 ml de FACS e adicionar uma dose de cocktail de anticorpos. </li><li> Criação de um painel de controles de cor única compensação para o citômetro de fluxo. Para cada fluorocromo a ser utilizado, misturar 50 ul de células não ligadas que tenham ficado fracção num tubo de 5 ml de FACS com 1 ul de anticorpo anti-CD4 conjugado com o fluorocromo correspondente. Lembre-se de configurar um controle sem manchas também. </li><li> Agitar em vórtice e incubar as amostras durante 30 min a 4 ° C. </li><li> Adicionam-se 5 ml de tampão de classificador a cada tubo e centrifugar durante 5 minutos a 300 xg, 4 ° C. </li><li> Para a fracção ligada, aspirar cuidadosamente o sobrenadante e ressuspender as células em 200 ul de buf classificadorfer. Transferir as células para um microtubo de 1,2 ml de FACS. Enxaguar o tubo com outro ul de tampão 200 e separador de piscina para o microtubo mesmo. Se aglomerados de células são evidentes, passar as células através de um filtro ^ M 50. </li><li> Para a fracção livre e controles de compensação, decantar ou aspirar o sobrenadante e ressuspender em tampão classificador 2 ml. </li><li> Configure o citômetro de fluxo, utilizando os controles de cor única compensação. Selecione lado dispersão de largura (SSC-W) como parâmetro adicional a ser gravado. </li><li> Analisar as amostras coradas utilizando uma sequência de sucessivas portas de inclusão para identificar células CD4 + ou CD8 +, como ilustrado nas Figuras 1 e 2. Para frações encadernados, coletar células como muitos como possível até um máximo de 2.000.000 total de eventos. Para fracções não ligadas, recolher 1.000.000 total de eventos. Manter a velocidade de aquisição em ou abaixo de 3000 eventos por segundo. </li><li> Usando as configurações da máquina mesmo, analisar as amostras de contagem de contas. Colete 10.000 total de eventos. &lt;/ Li&gt; <li> Guarde todos os dados como arquivos FCS. </li></ol> 5. Análise de Dados <ol><li> Analisar os arquivos de dados de FCS para as amostras de contagem de contas usando o software FlowJo. Dispersão trama para frente por FITC e definir uma porta aroung os colares de contas (ver Figuras 1 e 2). Determinar o número total de pérolas de contagem detectadas em cada amostra. Subtrair a partir do número total de eventos recolhidos para determinar o número de eventos celulares recolhidos. </li><li> Calcula-se o número total de todas as células em cada amostra, utilizando as equações descritas no Quadro 1. </li><li> Analisar os arquivos de dados para os FCS manchadas amostras fração associados e não associados. Defina-se uma sequência de sucessivas portas de inclusão para identificar + linfóide, do lado de dispersão de largura de eis-despejo, CD3 +, CD4 + ou CD8 +, células tetrâmero + T em cada uma das amostras tal como é ilustrado nas Figuras 1 e 2. </li><li> Multiplicar o número total de células na amostra por as frequências de cada um dos portões de inclusão usados ​​para definir CD4 + tetrâmero+ Ou CD8 + + tetrâmero células para calcular o número total de células T específicas do epítopo (Quadro 1). </li></ol>

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Não há conflitos de interesse declarados.

O pMHC tetrâmero método de enriquecimento baseado célula apresentada por este protocolo é uma ferramenta poderosa para estudar epítopo-específicas células T de endógenos repertórios de células T. O uso de tetrâmeros pMHC permite a detecção de células T específicas para epitopo diretamente dependente da capacidade dos seus TCR se ligar ligandos cognatos pMHC. O enriquecimento fornece um nível de sensibilidade de modo que as populações extremamente raros de células T específicas de antigénio pode ser ...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Reagente</strong</td&gt;<td<strong&gt; Vendedor</strong</td&gt;<td<strong&gt; Número de catálogo</strong</td&gt;</tr&gt;<tr&gt;<td&gt; PE ou APC conjugada pMHC tetrâmero (ou multimeros)</td&gt;<td&gt; Made pelo investigador, obtido a partir do núcleo do tetrâmero NIH, ou adquiridos de fontes comerciais</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-PE conjugados microesferas magnéticas</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-048-801</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-APC conjugados microesferas magnéticas</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-090-855</td&gt;</tr&gt;<tr&gt;<td&gt; Colunas LS magnéticos</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-042-401</td&gt;</tr&gt;<tr&gt;<td&gt; MidiMACS ou QuadroMACS ímã</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-042-302 ou 130-090-976</td&gt;</tr&gt;<tr&gt;<td&gt; Contagem de células contas</td&gt;<td&gt; Life Technologies</td&gt;<td&gt; PCB-100</td&gt;</tr&gt;</tbody&gt;</table

peptide:mhc tetramer-based enrichment of epitope-specific t cells

Uma necessidade básica para os investigadores que estudam a imunidade adaptativa com modelos experimentais in vivo é a capacidade de identificar as células T com base no seu receptor de células T de antigénio (TCR) especificidade. Muitos métodos indirectos estão disponíveis em que uma população a granel de células T é estimulada in vitro com um antigénio específico e células T específicas do epítopo são identificadas através da medição de uma resposta funcional, como a proliferação, a produção de citocinas, ou a expressão de marcadores de activação 1. No entanto, estes métodos apenas identificar as células T específicas para epitopo que exibem uma de muitas possíveis funções, e eles não são suficientemente sensíveis para detectar epítopos específicos de células T naive em frequências precursoras. Uma alternativa popular é o TCR transgénico modelo de transferência adoptiva, em que as células T monoclonais a partir de um ratinho transgénico TCR são semeadas em hospedeiros histocompatibilidade para criar um precursor população grande de células T específicas para epitopos que podem ser easily rastreados com o uso de um anticorpo marcador Congênicos 2,3. Embora poderoso, este método sofre de artefactos experimentais associados não fisiológicas da frequência de células T com especificidade para um único epítopo 4,5. Além disso, este sistema não pode ser utilizado para investigar a heterogeneidade funcional do epítopo específicos clones de células T dentro de uma população policlonal. A forma ideal para o estudo da imunidade adaptativa deve envolver a detecção directa do epítopo de células T específicas a partir do repertório de células T endógeno, utilizando um método que diferencie a especificidade TCR unicamente pela sua ligação ao péptido cognato: (pMHC) do MHC complexos. O uso de tetrâmeros pMHC e citometria de fluxo realiza esta 6, mas está limitado à detecção de populações de alta frequência de células T específicas para epitopo apenas encontrados a expansão induzida pelo antigénio seguinte clonal. Neste protocolo, descrevemos um método que coordena o uso de tetrâmeros pMHC e magnetic tecnologia de enriquecimento de células para permitir a detecção de frequências extremamente baixas células epítopo T específicas de tecidos de camundongos linfóides 3,7. Com esta técnica, pode-se rastrear exaustivamente inteiras epitopo-populações específicas de células T endógenas em ratinhos em todas as fases da resposta imune.

A Figura 1 mostra gráficos representativos de fluxo de citometria de pMHCII baço enriquecida tetrâmero e amostras de nódulos linfáticos de ratinhos ingénuos, enquanto que a Figura 2 mostra os dados representativos dos ratos anteriormente imunizados com o péptido relevante + CFA. Gating Serial remove autofluorescente e outros eventos indesejados a partir da análise das células T CD4 + populações de células T. A CD8 + da população de células T serve como um controlo interno...

Watch this Scientific Journal Video about Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells at JoVE.com

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

Este protocolo descreve o uso de péptido: MHC tetrâmeros e microesferas magnéticas para isolar populações de baixa frequência de células T específicas para epitopo e analisá-las por citometria de fluxo. Este método permite o estudo direto de endógenos populações de células T de interesse de<em&gt; In vivo</em&gt; Sistemas experimentais.

Péptido: Enriquecimento tetrâmero com base MHC das células T específicas para epitopos

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

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Immunology and Infection

17.8K visualizações.  Massachusetts General Hospital and Harvard Medical School. Este protocolo descreve o uso de péptido: MHC tetrâmeros e microesferas magnéticas para isolar populações de baixa frequência de células T específicas para epitopo e analisá-las por citometria de fluxo. Este método permite o estudo direto de endógenos populações de células T de interesse de In vivo Sistemas experimentais.

Vídeo: Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

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

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Geração de Células T reguladoras induzidas a partir de células T humanas primárias Naïve e Memória

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac modificação do sistema Transposon de células T primárias Humanos

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

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

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

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

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

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

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Geração de Células T humanas alo-específicos a partir de Sangue Periférico

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

As células T de enriquecimento de células automatizado de citomegalovírus específico-para aplicações clínicas usando o Sistema de captura de citocinas

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

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

As células T Capturar Bactérias por Transinfection das dendríticas

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

Produção de células CAR-T projetadas por CRISPR humanas

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with ...

Adoptive Transfer of P14 T-Cells into Tumor-Bearing Mice

Transferência Adotiva de Células T P14 em Camundongos Portadores de Tumor

Isolamento e caracterização de células T antígeno-específicas em pulmões de camundongos

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection.
As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

Autor em destaque: Protocolo otimizado para detecção de células T específicas de antígeno em pulmões de camundongos usando tetrâmeros

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC) tetramers.

Identificação de células T raras específicas de antígenos de pulmões de camundongos com peptídeos: tetrâmeros do complexo principal de histocompatibilidade

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune ...