Name: generation of human alloantigen-specific t cells from peripheral blood
Uploaded: 2014-11-21T15:00:00.000+00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Generation of Human Alloantigen-specific T Cells from Peripheral Blood at JoVE.com

The author would like to thank the NIH Tetramer Core Facility for tetramer production. The author would also like to thank E.D. O&#8217;Connor and K.E. Marquez at the UCSD Human Embryonic Stem Cell Core Facility flow cytometry laboratory for assistance in cell sorting. This work was funded by National Institutes of Health grant K08AI085039 (G.P.M.).

NOTA: Este protocolo requer a utilização de amostras de sangue periférico de voluntários humanos. Todas as pesquisas com seres humanos deve ser revisado e aprovado por um Estudos Humanos Institutional Review Board para assegurar a conformidade com a Declaração de Helsinque (2013) e do Seguro de Saúde Portabilidade e Responsabilidade Act de 1996. 1. Isolamento das células T a partir de Sangue Total <ol><li> Antes da partida, aquecer o meio de gradiente de densidade até à temperatura ambiente. Aliquota de 4 ml de meio de gradiente de densidade de 2-4 em tubos estéreis de 15 ml de centrífuga cónicos (1 tubo serão utilizados para cada 10 ml de volume total de sangue diluído). </li><li> Obter 10-20 ml de sangue em heparina spray de 2/1 de sódio revestido (verde-top) tubos de coleta de sangue venoso. Coletar amostras humanas sob a supervisão de pessoas treinadas e de acordo com um Conselho de Revisão Institucional protocolo aprovado. </li><li> Limpe a parte externa dos tubos com álcool a 70%. Remova cuidadosamente os tops da fiencheram tubos de coleta e decantar o sangue para um tubo de centrífuga de 50 ml estéril. </li><li> Adicionar 10 ml estéril tampão fosfato salino (PBS) a cada tubo de coleta de sangue. Recolher o PBS, adicionar ao sangue total decantado, e misture delicadamente. </li><li> Adicionar 25 ul t Human Enrichment celular Cocktail / 2 ml de volume total. Incubar à temperatura ambiente durante 20 min. </li><li> Utilizando uma pipeta de 10 mL, a camada de até 10 ml de 1: 1 sangue diluído suavemente no topo do meio de gradiente de densidade. Ter cuidado para não perturbar a superfície do meio de gradiente de densidade. </li><li> Centrifugar o sangue em camadas e meio de gradiente de densidade a 1200 xg durante 20 min a 20 o C. </li><li> Retire os tubos da centrifugadora, tendo cuidado para não perturbar a interface entre o meio de gradiente de densidade e a camada de leucócitos na interface entre o meio de gradiente de densidade e o plasma diluído. Usando uma pipeta de 5 ml, cuidadosamente a camada de leucócitos recolher e transferir para um novo 50 ml estéril banheira cónicae. </li><li> Adicionar PBS para perfazer o volume do PBL recolhidos para 50 mL e misturar suavemente. </li><li> Centrifugar a 600 xg durante 5 min a 20 o C. Decantar. </li><li> Ressuspender as células sedimentadas em 10 ml de citometria de fluxo estéril triagem tampão (esterilizada por filtração PBS contendo 1% de BSA). Amostra 10 ml de suspensão celular, diluir 1:10 (adicionar a 90 mL) azul de tripan para contar células usando um hemocitômetro (rendimento esperado 1-4 x 10 6 células / tubo de sangue total). <ol><li> Retirar alíquota de 1 ml, transferir para tubo de 5 ml, e manter em gelo para análise de células T não rotulados por tetrâmero. Mantenha suspensão de células no gelo. </li></ol></li></ol> 2. Enriquecimento magnética de células T alo-específicos <ol><li> Diluir aloantigénio pMHC tetrâmero a 1: 100 em tampão de tipo estéril. </li><li> Centrifugar a suspensão de células (9 ml do passo 1.11) a 600 xg durante 5 min a 20 o C. Decantar. </li><li> Adicionar 50 ul de aloantigénio diluída pMHC tetrâmero para sedimentadasAs células (até 10 7 células). Misture por pipetagem suave. Transferir para um tubo esterilizado de 5 ml. Incubar durante 30 minutos à temperatura ambiente. </li><li> Adicione 2 ml tipo buffer. Centrifugar a 600 xg durante 5 min a 20 o C. Decantar. </li><li> Ressuspender as células em 100 ul tampão tipo. Adicionar 10 ul de coquetel Biotina Selecção e incubar durante 15 min à temperatura ambiente. </li><li> Adicionar 5 uL Magnética Nanopartículas e incubar durante 10 min à temperatura ambiente. </li><li> Adicionar 2,5 ml espécie de tampão e misture delicadamente por pipetagem. Remover alíquota de 100 ul, transferir para tubo de 5 ml, e manter em gelo para análise de células de pré-enriquecimento. </li><li> Colocar o tubo contendo 5 ml da suspensão de células no íman de separação de células. A tampa deve ser solta em cima do tubo. Incubar durante 5 min à temperatura ambiente. </li><li> Com cuidado, retire a tampa do tubo. Segurando o tubo e um ímã em conjunto, decantar o conteúdo do tubo para um novo tubo de 5 ml. Não bata nem sacuda o conteúdo do tubo pararemover as últimas gotas de líquido. </li><li> Retirar o tubo do ímã. Adicionar 2 ml de tampão tipo frio e misture delicadamente por pipetagem. Amostra de 10 uL da suspensão de células, diluída 1: 2 (para adicionar 10 ul) de azul de tripano a contagem de células utilizando um hemocitómetro. </li></ol> 3. Preparação de células T para uma única célula Citometria de Fluxo Seleção celular <ol><li> Adicionar tampão tipo frio em todos os tubos de células (tetrâmero marcado, não-enriquecido, e enriquecido com rótulo tetrâmero marcado com tetrâmero) para trazer volume para 3 ml. </li><li> Centrifugar a 600 xg durante 5 min a 4 ° C e tampão de decantar para recuperar as células. </li><li> Ressuspender as células peletizadas em 25 mL de tampão tipo frio. Adicionar 5 uL de bloco Fc humana. Incubar em gelo durante 20 min. </li><li> Preparar anticorpos para triagem de citometria de fluxo. Misture 15 ul de tampão tipo, 15 ul anti-CD5, 15 ul anti-CD14, e 15 ul anti-CD19. </li><li> Adicionar 20 ul de mistura de anticorpo a cada tubo de células. Incubar em gelo durante 20 min. </li><li> Adicionar 2 ml de tampão de tipo frio a cada tubo. </li><li> Centrifugar a 600 xg durante 5 min a 4 ° C e tampão de decantar para recuperar as células. </li><li> Ressuspender as células a uma concentração de 1 x 10 -2 6 células / ml em tampão tipo. </li><li> Alíquotas de 100 ul de t meio de cultura de células humanas (DMEM Iscove suplementado com Glutamax 2 mM, HEPES 10 mM, 50 ug / ml de gentamicina, 50 uM de 2-mercaptoetanol, 10% de soro AB humano inactivado pelo calor, e 2,5 ng ml humana / recombinante IL-2) para cada poço de uma placa redonda de 96 poços de cultura de células de fundo. Manter em gelo. </li></ol> 4. Isolamento de células marcadas com tetrâmero T por uma única célula Citometria de Fluxo Seleção <ol><li> Estabelecer citometria de fluxo para identificar os parâmetros de propagação aloantigénio-pMHC tetrâmero células T marcados (Figura 2.a). Use a fração tetramer pré-enriquecimento marcado para estabelecer gating estratégias para eliminar dobletos, portão em linfócitos, excluir CD19 expressando as células B e CD14expressando monócitos, e identificar as células T por expressão CD5. Utilizar a amostra não marcada como um tetrâmero com fluorescência menos um controlo para estabelecer o gating para identificação de células T de ligação ao tetrâmero (com 0% de CD5 + CD14 - CD19 - células a partir da amostra não marcada pelo tetrâmero deve cair dentro deste portão) . NOTA: Gating estratégias que não são adequadamente rigorosas irá resultar no isolamento de células não-T ou células T que não são antigénio específico, enquanto as estratégias gating excessivamente restritivas vai reduzir o número de células T isoladas. </li><li> Programar os parâmetros da placa para o tipo de célula única. Direcionar o classificador para colocar um CD5 + CD14 - CD19 - tetrâmero + célula em cada poço. Direcione a placa criada para conter um controle negativo (ausência de células classificadas para o bem) e um controle positivo (100 CD5 + CD14 - CD19 - tetramer - células) em cada linha (Figura 2.B </strong&gt;). </li><li> Utilizando a citometria de fluxo estabelecida estratégia gating e configuração da placa, isolar as células-T de ligação de tetrâmero individuais directamente para a placa de 96 poços utilizando um classificador de células por citometria de fluxo. </li></ol> 5. Cultura e Expansão de clones de células T alo-específicas <ol><li> Após a conclusão da separação de células, a placa de centrifugar a 600 xg durante 5 min a 20 o C. Colocar a placa de cultura a 37 ° C num 6% incubadora de CO 2. </li><li> Vortex estimuladores microesferas anti-CD3 / anti-CD28 durante 30 seg para ressuspender as esferas. </li><li> Calcular o volume de grânulos activadores necessários para a estimulação de células T recolhidas (0,5 ul de microesferas de activadores / poço). Transferir volume calculado de contas estimulador para um tubo estéril de 5 ml. Adicionar 1 ml de meio de cultura de célula T humana e vortex. </li><li> Coloque tubo com suspensão microbead em ímã. A tampa deve ser solta em cima do tubo. Incubar durante 2 min à temperatura ambiente. </li><li> Retire com cuidadoda tampa do tubo. Segurando o tubo e um ímã em conjunto, o conteúdo do tubo decante para um novo tubo de 5 ml. Não bata nem sacuda o conteúdo do tubo. </li><li> Retirar o tubo do ímã. Adicionar meio de cultura de células T humanas (100 / ul médias 0,5 ul de microesferas inicialmente adicionada). </li><li> Aliquota de 100 ul de suspensão de activador do grânulo para cada poço da placa de 96 poços. Incubar a placa de cultura a 37 ° C em 6% de CO 2 incubadora. </li><li> Monitorar o crescimento celular diariamente pelo exame microscópico. Os pequenos agregados de células em proliferação podem ser observadas ao microscópio após 5-7 dias de cultura (Figura 3.A). NOTA: A visualização das culturas de células T, neste ponto pode ser difícil, e a ausência de agrupamentos de células observáveis, nesta fase, pode não ser indicativa de uma falta de crescimento de células T. </li><li> Aos 14 dias após o isolamento das células, culturas para alimentação animal, removendo cuidadosamente 100 ul de meio para fora do topo da cultura e substitua com 100 μ; L fresco humano T meio de cultura celular. Continuar a incubação placa de cultura a 37 ° C em 6% de CO 2 incubadora. </li><li> Monitorizar o crescimento celular por dia por exame microscópico e avaliação da cor do meio de cultura. Grandes grupos de células deve ser visível microscopicamente 2 - 3 dias após a mudança do meio. Macroscopicamente, pelotas de células deve tornar-se visível durante os 14 dias seguintes à mudança de meio. </li><li> Aos 28 dias após o isolamento de células, identificar clones que crescem através de exame microscópico. Transferir o volume de 200 ul das crescimento positivo de 96 poços da placa de culturas para poços individuais de uma placa de 48 poços de cultura de tecidos contendo 200 ul de meio de cultura de célula T humana. </li><li> Adicionar 100 ul contendo 12,5 ul de microesferas anti-CD3 / anti-CD28 estimuladores (preparada tal como descrito nas secções 5,2-5,6). Incubar a placa de cultura a 37 ° C em 6% de CO 2 incubadora. </li><li> Monitorar o crescimento cultura diariamente por ex microscópicaaminação e avaliação da cor do meio de cultura. Quando a cultura de células T atinge&gt; 2x10 6 / ml (tipicamente 3-5 dias após re-estimulação, pode ser estimado pelo dia em que os meios de comunicação começa a transformá amarelo-palha), cultura da transferência para um poço de uma placa de cultura de tecido de 24 poços e adicionar 500 mL meio humana de células T. </li><li> No dia 14/10 siga re-estimulação, coletar 200 mL de cultura de células T para avaliar aloantígeno especificidade análise por citometria de fluxo de pMHC tetramer ligação (utilizando a estratégia de rotulagem e gating descrito nas secções 3 e 4.1). </li></ol> 6. longo prazo Re-estimulação e Cultura de clones de células T <ol><li> Continuamente re-estimular e expandir culturas clonais de células T com a especificidade desejada podem ser a cada 14 dias. Recolhe culturas de células T para um tubo de 5 ml estéril no dia 14 após a última anti-CD3 / CD28 estimulação micropérola. Combinação de várias cavidades do mesmo clone de células T num único tubo, até um volume de 2,5 ml. </li&gt; <li> Adicionar meio para levar o volume final para 2,5 ml e misturar suavemente por pipetagem. </li><li> Colocar o tubo de 5 ml contendo a cultura de células T para o íman de separação de células para remover velhos microesferas a partir da cultura. A tampa deve ser solta em cima do tubo. Incubar durante 5 min à temperatura ambiente. </li><li> Com cuidado, retire a tampa do tubo. Segurando o tubo e em conjunto íman, decanta-se a suspensão de células para um novo tubo de 5 ml. Não bata nem sacuda o conteúdo do tubo para remover as últimas gotas de líquido. </li><li> Adicionar meio para levar o volume final a 4 ml. Amostra de 10 mL para contagem de células usando um hemocitômetro. </li><li> Recuperar as células T através de centrifugação a 600 xg durante 5 min à temperatura ambiente. </li><li> Decantar o sobrenadante. As células em 10 6 células / ml T humanas meio de cultura de células T Ressuspender. Transferir 1 ml de partes alíquotas para cada poço de uma placa de cultura de tecidos de 24 cavidades. </li><li> Re-estimular as células T através da adição de 100 ul contendo meios de 12,5 ul de estimulador anti-CD3 / amicrobeads nti-CD28 (conforme descrito nas seções 5,2-5,6). Incubar a 37 ° C em 6% de CO 2 incubadora. </li></ol>

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The authors declare no competing financial interests.

T cell alloreactivity is a long-studied and clinically-relevant phenomenon. The robust proliferative and effector responses of T cells to allogeneic stimulation has enabled extensive analyses of human T cell responses in vitro through relatively straightforward mixed lymphocyte reactions of peripheral blood T cells against inactivated allogeneic cells. However, these primary alloreactive T cell responses are oligoclonal, comprised of a large number of individual T cells responding to specific alloantigens. This ...

Os linfócitos T são componentes críticos do sistema imunitário adaptativo. As células T são responsáveis ​​não só para mediar directamente respostas imunitárias protectoras para agentes patogénicos através de uma variedade de mecanismos efectores, mas também manter activamente auto-tolerância imunológica e dirigir as respostas de outras células do sistema imunológico. Estas funções são dirigidos através de um número de sinais integrados, incluindo o receptor de células T (TCR) de ligação, citocinas e quimiocinas, e metabolitos 1. Destes sinais, o TCR é de particular importância, uma vez que proporciona a especificidade característica que define o papel de células T na imunidade adaptativa. Um TCR interage com antigénios peptídicos lineares apresentados pelo MHC (HLA ortólogo humano) moléculas (complexos pMHC) de um modo altamente específico e sensível para fornecer os sinais que dão início a função efectora de células T. Os parâmetros bioquímicos das interacções TCR com ligandos pMHC proporcionam não só a especificidade para o tativação celular, mas também tem um impacto qualitativo sobre a função das células T posterior 2. Assim, estudar a função das células T exige frequentemente a examinar as respostas de células T com especificidade antigénica clonais definido. O compartimento da célula T humana, contendo cerca de 10 12 células T αβ, contém uma estimativa de 10 julho-10 agosto αβTCRs distintas 3-4. Este repertório diversificado oferece oportunidade para o reconhecimento da vasta gama de peptídeos de potenciais patógenos que exigiria uma resposta das células T para a imunidade protetora. Estima-se que a frequência de células T de responder a um determinado antigénio estranho apresentado por auto-MHC é da ordem de 10 -4 - -7 10 na ausência de resposta imune antes que o antigénio 5. O repertório de células T naive é moldada por selecção tímica para assegurar a capacidade de reconhecer auto-MHC que apresenta antigénios peptídicos e limite reagemivity contra antigénios auto-péptido, a maximização da utilidade potencial para mediar a imunidade protectora 2. No entanto, em violação da presente concebido reactividade, uma relativamente grande frequência, 10 -3 - -4 10, de células T a partir de indivíduos imunologicamente naive respondem à estimulação com células alogénicas, que reconhece ambas as moléculas MHC estranhos, bem como os peptídeos endógenos que apresentem 6. O reconhecimento de ligandos pMHC alogénicos é estruturalmente semelhante ao do reconhecimento de antigénios estranhos apresentados pelas auto-MHC; o TCR faz interacções bioquímicas críticas, tanto com a molécula de MHC alogénico, bem como o péptido apresentado 7. A natureza robusta da resposta das células T para estimulação resulta alogénicas a partir da diversidade de complexos pMHC presente na superfície de células alogénicas 8. Estima-se que cada MHC apresenta cerca de 2 x 10 4 diferentes antigénios peptídicos endógena 9. Este breadth de resposta à estimulação alogénica é um aspecto importante da patologia clinicamente relevantes, tais como a rejeição de aloenxertos ou a doença do enxerto versus hospedeiro (GVHD), que resulta a partir de células T alloreactivity. Estudo de células T respostas alorreativas humanos tradicionalmente invocado examinar as respostas policlonais de células T naive após a estimulação com células alogênicas. Estimulação repetida com a mesma linha de célula alogénica combinado com análises de diluição limitante é capaz de gerar células T clonais com reconhecimento definido alogénica de HLA de 10. No entanto, esta abordagem é problemática para examinar respostas aos ligandos alogénicos pMHC individuais, como o repertório grande e diverso de complexos pMHC endógeno presente para um dado alogénica de HLA estimula um amplo repertório de células T. Esta estimulação população a granel e de diluição limitante abordagem exigiria o rastreio de grandes números de clones para isolar as células T com a desejada Reactividade contra um único ligando pMHC. Além disso, a frequência de células T que respondem a uma célula alogénica ligando pMHC individuais é relativamente baixo entre as populações de células T ingénuas, o que apresenta uma barreira eficiente para a geração de clones de células T humanas que respondem a um dado antigénio. Identificação e isolamento de células T específicas de antigénios a partir de populações policlonais têm sido activado pelo desenvolvimento de marcado com fluoróforo pMHC multímeros 11. Esta abordagem utiliza antigénios peptídicos específicos carregados em moléculas de MHC recombinantes solúveis, biotinilados que são marcados por ligação a um fluoroforo marcado com estreptavidina. Multimerização de pMHC aumenta a avidez, compensando a afinidade intrínseca baixa (M) de TCR para ligantes pMHC solúveis. As células marcadas podem ser identificadas e isoladas por citometria de fluxo. No entanto, esta abordagem é ainda limitada pela baixa frequência de células T específicas do antigénio entre as populações de células T naive, que são tipicamenteordens de grandeza menor do que o limite de identificação e quantificação precisas sobre a maioria das citómetros de fluxo. Para resolver esta limitação, um método de rotulagem pMHC tetrâmero e subsequente enriquecimento esfera magnética para células marcadas com tetrâmero foi desenvolvida 12. Este método tem demonstrado uma detecção fiável, enumeração e isolamento de células específicas para o antigénio T de baixa frequência. Este protocolo descreve um protocolo eficaz para a geração de clones de células T humanas que respondem especificamente a ligandos alogénicos pMHC individuais. O protocolo aplica pMHC (HLA) de rotulagem e enriquecimento multímero para o isolamento de células T humanas específicas do aloantigénio com citometria de fluxo de células de triagem e um método robusto para a cultura in vitro de células T humanas, para permitir a produção de clones de células T a partir de células separadas individuais ( Resumo na Figura 1).

<table><tbody><tr><td>Sodium heparin venous blood collection tube 16 x 100 mm</td><td>Becton, Dickenson and Company</td><td>366480</td><td></td></tr><tr><td>Lymphoprep</td><td>Stemcell Technologies</td><td>7801</td><td></td></tr><tr><td>Rosette Sep Human T Cell Enrichment Kit</td><td>Stemcell Technologies</td><td>15061</td><td></td></tr><tr><td>Dulbecco&#039;s PBS, 1x without Ca or Mg</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6635</td><td></td></tr><tr><td>Fluorophore-labeled pMHC tetramer</td><td>NIH Tetramer Facility</td><td>NA</td><td></td></tr><tr><td>EasySep Biotin Selection Kit</td><td>Stemcell Technologies</td><td>18553</td><td></td></tr><tr><td>EasySep Selection magnet</td><td>Stemcell Technologies</td><td>18000</td><td></td></tr><tr><td>TruStain FcX Human Fc blocking solution</td><td>Biolegend</td><td>422301</td><td></td></tr><tr><td>Anti-CD5 PE-Cy7 (clone UCHT2)</td><td>Biolegend</td><td>300621</td><td></td></tr><tr><td>Anti-CD14 FITC (clone HCD14)</td><td>Biolegend</td><td>325603</td><td></td></tr><tr><td>Anti-CD19 FITC (clone HIB19)</td><td>Biolegend</td><td>302205</td><td></td></tr><tr><td>Iscove&#039;s DMEM, without b-ME or L-glutamine</td><td>Corning</td><td>15-016-CV</td><td></td></tr><tr><td>HEPES</td><td>Corning</td><td>25-060-CI</td><td></td></tr><tr><td>b-Mercaptoethanol&amp;nbsp;</td><td>Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>Gentamicin sulfate (50 mg/ml)</td><td>Omega Scientific</td><td>GT-50</td><td></td></tr><tr><td>Human AB serum, male donor</td><td>Omega Scientific</td><td>HS-30</td><td></td></tr><tr><td>Recombinant human IL-2</td><td>Peprotech</td><td>AF 200-02</td><td></td></tr><tr><td>Dynabeads Human T-Activator CD3/CD28</td><td>Life Technologies</td><td>11131D</td><td></td></tr><tr><td>Media</td><td></td><td></td><td></td></tr><tr><td>Cell sorting buffer</td><td></td><td></td><td></td></tr><tr><td>PBS, pH 7.4</td><td>1 L</td><td></td><td></td></tr><tr><td>BSA</td><td>10 g</td><td></td><td></td></tr><tr><td>EDTA (0.5 M)</td><td>2 ml</td><td></td><td></td></tr><tr><td>Human T Cell Culture Medium</td><td></td><td></td><td></td></tr><tr><td>Iscove&#039;s DMEM</td><td>351.6 ml</td><td></td><td></td></tr><tr><td>Heat-inactivated human AB serum</td><td>40 ml</td><td></td><td></td></tr><tr><td>HEPES (1 M)</td><td>4 ml</td><td></td><td></td></tr><tr><td>Glutamax (100x)</td><td>4 ml</td><td></td><td></td></tr><tr><td>Gentamicin (50 mg/ml)</td><td>0.4 ml</td><td></td><td></td></tr><tr><td>b-mercaptoethanol (14.3 M)</td><td>1.4 ml</td><td></td><td></td></tr><tr><td>Recombinant human IL-2 (1 mg/ml)</td><td>1 ml</td><td></td><td></td></tr></tbody></table>

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.

Este protocolo descreve a geração de culturas de células T humanas clonal com aloantigénio especificidade definida por meio de um cordão de enriquecimento magnética e fluxo de uma única célula citometria de triagem estratégia. A Figura 1 apresenta um esboço do processo. <img alt="A Figura 1" src="/files/ftp_upload/52257/52257fig1highres.jpg" /> Figura 1: Vista geral Protocolo O protocolo aqui descrito prop...

Watch this Scientific Journal Video about Generation of Human Alloantigen-specific T Cells from Peripheral Blood at JoVE.com

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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 ...

generation-of-human-alloantigen-specific-t-cells-from-peripheral-blood

Research

JoVE Journal

Immunology and Infection

12.9K visualizações.  University of California, San Diego. 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.

Vídeo: Generation of Human Alloantigen-specific T Cells from Peripheral Blood

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

Indução de aloantígenos específicos de anergia em células mononucleares do sangue periférico por Estimulação com aloantígenos Co-estimulatórias bloqueio de sinal

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

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&#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 ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Geração de Integração-livres induzidas células-tronco pluripotentes a partir de células mononucleares de sangue periférico humano Usando epissï¿½ica Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Biologia do Desenvolvimento

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

Usando células-tronco pluripotentes induzidas pelo homem para a geração de células T específicas de antígeno tumoral

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Pesquisa do câncer

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Geração de humanos CD40-activated células B

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Biologia

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

Isolamento e<em&gt; Ex Vivo</em&gt; Cultura de Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; Células T, γδ um Extrathymic αβT de células progenitoras

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

Gerando<em&gt; De Novo</em&gt; Antígeno-específica T Humano receptores celulares por Transdução retroviral de Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Geração de células dendríticas derivadas de monócitos humanos a partir de sangue inteiro

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Geração dos anticorpos monoclonais humanos discriminativa de rara B antígeno-específicas células circulantes no sangue

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Generation of Human Regulatory T Cell Clones

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as &#8220;Treg&#8221; therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4+CD25+ T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based ...

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

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