1. Isolamento de PBMC <ol><li> Coleta de sangue (7,5-8,0 ml) em um BD Vacutainer CPT Tubo preparação celular com heparina sódica. O tubo contém um anticoagulante heparina sódica e um fluido de densidade Ficoll-Hypaque, além de uma barreira de gel de poliéster, que separa os dois líquidos. Tubo de centrifugação / amostra de sangue em temperatura ambiente (18 ° C a 25 ° C) em um rotor horizontal (swing out-cabeça) por 20 min a 1800 x g. Interruptor centrífuga freios off. </li><li> Após a centrifugação, a seqüência de camadas ocorre da seguinte forma (visto de cima para baixo): um plasma) - b) células mononucleares do sangue periférico (PBMC) e plaquetas - c) solução de densidade - d) gel de poliéster - e) granulócitos - f) glóbulos vermelhos (Fig. 1). </li><li> Coletar uma fração da camada de plasma, deixando 5 a 10 mm de plasma acima da interfase sem perturbar a camada de células. O plasma pode ser usado para a cultura (seção 2.5). </li><li> Colheita da fração enriquecida (PBMC) na interfase com uma pipeta e transferir para um tubo de 15 ml. </li><li> Lave o PBMC com 10 ml de tampão fosfato (PBS), invertendo o tubo 5 vezes, e então centrifugar por 5 min a 400 x g. </li><li> Repita os passos de lavagem duas vezes, e, em seguida, ressuspender o sedimento celular em 5 ml de PBS. Determinar o número de células. Normalmente, 1,3 x10 6 células são recuperados a partir de 1 ml de sangue total. </li><li> Determinar a freqüência e fenótipo de células T γδ em PBMC por citometria de fluxo (seção 3) (Fig. 2). </li></ol> 2. Expansão de células T γδ <ol><li> Centrífuga suspensões de células em 15 ml tubos cônicos por 5 min a 400 x g à temperatura ambiente, e descartar o sobrenadante. </li><li> Prepare meio de cultura (CM), acrescentando IL-2 humana (IL-2) e zoledronato (Zometa) para concentrações finais de 1000 UI / ml e 5 mM, respectivamente. ALyS203 (Cell Science &amp; Technology Institute) ou Optmizer (Invitrogen) o suporte a expansão de células γδ boa T (mais informações em referências 6 e 9). Zometa é fornecido na forma líquida (4 frasco mg/5-ml). Para preparar uma solução de M 5, adicione 50 ul de Zometa para 30 ml de meio de cultura. </li><li> Ressuspender pellet celular em meio de cultura e ajustar a 1x10 6 células / ml. </li><li> Ml pipeta 1 de CM contendo 1x10 6 células em cada poço de uma placa de 24 poços. De grande escala culturas, as células podem ser semeados em 0,5 x 10 6 células / cm 2 de acordo com as áreas de superfície dos poços da placa, prato, ou balão. </li><li> Adicionar plasma autólogo (seção 1.3), agrupados humano AB soros, ou FCS para que seja aproximadamente 10% do volume da cultura (100 l para cada poço de uma placa de 24 poços). Coloque as placas em um umidificado 37 ° C, 5% incubadora de CO 2 por 24-48 horas. </li><li> Manter a cultura, a uma densidade de células de 0,5-2 x 10 6 células / ml. Adicionar meio fresco contendo IL-2 humana (1000 UI / ml) apenas (sem Zometa) a cada 2-3 dias, ea transferência de células cultivadas em novos poços ou frascos, se necessário, de acordo com o grau de proliferação celular (Fig. 3). Fornecimento de plasma ou soro ao meio de modo que a concentração sérica pode ser mantida pelo menos 1%. </li><li> Células colheita no dia 14/12 e determinar a freqüência de fenótipo, e as funções de células T γδ por citometria de fluxo (veja abaixo). </li></ol> 3. Análise fenotípica por citometria de fluxo <ol><li> Transferência de 200 amostras l contendo 2 x 10 5 células para fluorescência-activated cell sorting (FACS) tubos. </li><li> Adicionar 2 ml de frio PBS e centrifugar por 5 min a 400 x g. Então, voltar a suspender as pelotas em 50 mL tampão FACS (PBS + 1% FCS azida de sódio + 0,1%). Adicionar 5 mL de cada anticorpos para as amostras (os anticorpos monoclonais estão listados na Tabela 1). </li><li> Incubar no gelo no escuro por 20 min. </li><li> Adicionar 2 ml de tampão FACS para cada amostra, e depois de vórtice. Amostras de centrífuga por 5 min a 400 x g a 4 ° C. Decantar cuidadosamente o sobrenadante. </li><li> Ressuspender as células em 300 mL de buffer FACS e vortex. Analisar amostras em um citômetro de fluxo (Fig. 2). </li></ol> 4. IFN-γ ensaio de produção de 10 <ol><li> Um dia antes do ensaio, preparar células estimulador através da cultura 05/03 x10 5 células Daudi / ml em meio RPMI 1640, mais 10% SFB (RPMI-10) durante a noite com Zometa (5 mM) (doravante designado Z-Daudi). </li><li> Coletar Z-Daudi e ressuspender em RPMI-10 em 2x10 6 células / ml. Adicionar 100 ul de Z-Daudi (2x10 5) a cada poço de uma placa de fundo redondo de 96 poços. </li><li> Prepare γδ células T em 2x10 6 células / ml em RPMI-10 contendo Brefeldin A a 20 mg / ml. Transferência de 100 l da suspensão de células T γδ (2x10 5) a cada poço contendo Z-Daudi células ou para controlar poços (100 ul de RPMI-10 apenas, ou RPMI-10 com 20 ng / ml de forbol 12-miristato 13-acetato[PMA] mais 2 mg / ml de ionomicina). </li><li> Mix pipetando cima e para baixo várias vezes. Incubar durante 4 horas a 37 ° C, 5% CO 2 incubadora. </li><li> Centrifugar a placa por 5 min a 400 x g a 4 ° C e ressuspender o pellets em 200 mL de frio PBS. </li><li> Transferir as amostras para tubos FACS. Adicionar 4 ml de frio PBS e centrifugar os tubos por 5 min a 400 x g a 4 ° C. </li><li> Volte a suspender as pelotas em 50 ul de tampão FACS com FITC-conjugado anti-TCRVγ9 (5 mL) e PE/Cy5-conjugated anti-CD3 mAb (2,5 mL). Incube ao abrigo da luz por 15 min em temperatura ambiente. </li><li> Adicionar 100 ul de IntraPrep reagente 1 e incubar por 15 min em temperatura ambiente. Adicionar 4 ml de PBS a cada tubo e centrifugar por 5 min a 400 x g à temperatura ambiente. </li><li> Retire o sobrenadante por aspiração e adicione 100 ml de IntraPrep dois reagentes. Incubar por 5 min à temperatura ambiente sem agitação. </li><li> Adicionar 5 mL da PE-conjugado anti-IFN-γ mAb ao tubo de ensaio. Incube ao abrigo da luz por 15 min em temperatura ambiente. </li><li> Adicionar 4 ml de PBS a cada tubo e centrifugar por 5 min a 400 x g à temperatura ambiente. Retire o sobrenadante por aspiração e ressuspender o pellet celular em 0,5 ml de tampão FACS. </li><li> Analisar as células por citometria de fluxo. Portão de CD3 + + TCRVγ9 células e examinar a expressão de IFN-γ (Fig. 4). </li></ol> 5. Resultados representativos: É importante para determinar a porcentagem de células T γδ em PBMC no início da cultura. Como mostrado na figura. 2 A, a porcentagem de células CD3 + + TCRVγ9 células T γδ em PBMC foi de 1,6% no dia 0. As populações dominantes eram CD27 + CD45RA + CD27 + ingênuo ou CD45RA - fenótipos de memória central. Quando as células T γδ foram eficientemente estimulados, eles formaram clusters no dia 05/03 (Fig. 3 A e B). Quando a formação do cluster foi adiada, o crescimento de outros tipos de células, tais como CD4 + ou CD8 + células T αβ ou células NK poderia dominar o crescimento de células T γδ (Fig. 3 C e D). Após 14 dias de cultura, a freqüência de células T γδ aumentou para mais de 93,8% das células cultivadas em culturas de células γδ sucesso T (Fig. 2 E). A cultura de células T γδ NKG2D upregulated CD69 e expressão (Fig. 2 G e H). Eles exibido CD27 - CD45RA - fenótipo de memória efetoras (Fig. 2 F). As funções das células T γδ foram avaliados em relação à produção de citocinas e citotoxicidade. IFN-γ o intracelular coloração demonstraram que as células T γδ IFN-γ produzido em resposta a PMA / ionomicina tratamento ou Z-Daudi células que IPP acumulado após o tratamento zoledronato (Fig. 4). Estes resultados indicam que zoledronato pode eficientemente estimular e expandir funcional das células T γδ. <table border="1"><tbody><tr><td> tubo </td><td> FITC </td><td> PE </td><td> ECD </td><td> PE/Cy5 </td></tr><tr><td> 1 </td><td> CD3 </td><td> CD19 </td><td> CD45 </td><td> CD14 </td></tr><tr><td> 2 </td><td> CD3 </td><td> TCRαβ </td><td> CD4 </td><td> CD8 </td></tr><tr><td> 3 </td><td> CD3 </td><td></td><td></td><td> CD56 </td></tr><tr><td> 4 </td><td> TCR Vγ9 </td><td> TCRαβ </td><td> CD45 </td><td> CD3 </td></tr><tr><td> 5 </td><td> TCR Vγ9 </td><td> NKG2D </td><td></td><td></td></tr><tr><td> 6 </td><td> TCR Vγ9 </td><td> CD69 </td><td></td><td></td></tr><tr><td> 7 </td><td> TCR Vγ9 </td><td> camundongo IgG1 </td><td></td><td></td></tr><tr><td> 8 </td><td> TCR Vγ9 </td><td></td><td> CD45RA </td><td> CD27 </td></tr><tr><td> 9 </td><td> TCR Vγ9 </td><td></td><td> camundongo IgG1 </td><td> camundongo IgG1 </td></tr></tbody></table> Tabela 1. Anticorpos monoclonais utilizados no multicolor de coloração de células T γδ. Um exemplo da análise fenotípica de células T γδ realizados em nosso laboratório é mostrada na figura. 2. <img src="/files/ftp_upload/3182/3182fig1.jpg" alt="Figura 1"/> Figura 1. Separação de PBMC. Sangue (7,5-8,0 ml) é desenhada em um BD Vacutainer CPT Tubo preparação celular com heparina sódica e directamente centrifugada por 20 min a 1800 x g. Após a centrifugação, as camadas, resultando como visto de cima para bottom: a Plasma) - b) PBMC e plaquetas - solução Densidade c) - d) gel de poliéster - e) Granulócitos - f) Os glóbulos vermelhos. <img src="/files/ftp_upload/3182/3182fig2.jpg" alt="Figura 2"/> Figura 2. Fenótipo de superfície típica de células T γδ. PBMC foram estimuladas com zoledronato e IL-2 por 14 dias. Células foram marcadas com FITC-rotuladas anti-TCR Vγ9 e PE/Cy5-labeled anti-CD3 para monitorar a expansão de células T γδ (A e E). As células T γδ foram identificados por sua expressão de TCRVγ9, e sua expressão de CD27 e CD45RA (B e F), NKG2D (C e G), ou CD69 (D e H) foi examinada. <img src="/files/ftp_upload/3182/3182fig3.jpg" alt="Figura 3"/> Figura 3. Representante culturas de células T γδ. PBMC foram estimuladas com IL-2 (1.000 UI / ml) e zoledronato (5 mM). Campos representativos são mostrados (IX71 microscópio invertido [Olympus] x 200). Aglomerados e agregados de células T γδ podem ser observados no dia 3 (A) e dia 5 (B), quando as células T γδ expansão com sucesso. Em contraste, não aglomerados ou agregados foram observados quando o crescimento das células T γδ não era adequado (C e D). <img src="/files/ftp_upload/3182/3182fig4.jpg" alt="Figura 4"/> Figura 4. IFN γ-produção. As células T γδ foram incubadas com RPMI-10 apenas (A) ou PMA / ionomicina (B) ou Z-Daudi (C) por 4 horas. Em primeiro lugar, a expressão de superfície de TCRVγ9 estava manchada e IFN-γ, em seguida, a produção foi examinado por intracelular IFN-γ coloração. <img src="/files/ftp_upload/3182/3182fig5.jpg" alt="Figura 5"/> Figura 5. Cinética da cultura de células T γδ. (A) o número absoluto de células em cultura, (B) porcentagem de células T γδ, e (C) número absoluto de células T γδ nos pontos de tempo indicado.

<ol>
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O método aqui apresentado permite a expansão eficiente de células T γδ do PBMC. células γδ T ativadas e se expandiu por zoledronato e IL-2 completa desenvolver funções efetoras, refletido pela produção de citocinas e citotoxicidade. Tem sido relatado que o pirofosfato bromohydrin sintéticos phosphoantigens (BrHPP) e 2-metil-3-butenyl-1-pirofosfato (2M3B1PP) também expandir as células T γδ, no entanto, eles não estão disponíveis comercialmente. Em contraste, zoledronato já está licenciado para aplic...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> ZOMETA </td><td> Novartis Pharma K. K </td><td></td><td> zoledronato </td></tr><tr><td> Proleukin </td><td> Novartis Pharmaceuticals </td><td></td><td> IL-2 recombinante humana </td></tr><tr><td> BD Vacutainer CPT Tubo preparação celular com heparina sódica </td><td> BD </td><td> 362753 </td><td></td></tr><tr><td> RPMI1640 </td><td> Invitrogen </td><td> 21870-076 </td><td></td></tr><tr><td> ALyS203 médio </td><td> Cell Science &amp; Technology Institute </td><td> 0301-7 </td><td></td></tr><tr><td> Optmizer </td><td> Invitrogen </td><td> 0080022SA </td><td></td></tr><tr><td> A brefeldin </td><td> Sigma </td><td> B5936-200UL </td><td></td></tr><tr><td> forbol 12-miristato 13-acetato (PMA) </td><td> Sigma </td><td> P1585-1MG </td><td></td></tr><tr><td> ionomicina </td><td> Sigma </td><td> 13909-1ml </td><td></td></tr><tr><td> IntraPrep </td><td> BECKMAN COULTER </td><td> A07803 </td><td></td></tr><tr><td> anti-CD3 humano-FITC ou PE/Cy5 </td><td> BECKMAN COULTER </td><td> A07746 FITC A07749 PE/Cy5 </td><td></td></tr><tr><td> anti-humano CD4-ECD </td><td> BECKMAN COULTER </td><td> 6604727 </td><td></td></tr><tr><td> anti-humano CD8-PE/Cy5 </td><td> BECKMAN COULTER </td><td> 6607011 </td><td></td></tr><tr><td> anti-humano CD14-PE/Cy5 </td><td> BECKMAN COULTER </td><td> A07765 </td><td></td></tr><tr><td> anti-CD19 humano-PE </td><td> BECKMAN COULTER </td><td> A07769 </td><td></td></tr><tr><td> anti-CD45 humano-ECD </td><td> BECKMAN COULTER </td><td> A07784 </td><td></td></tr><tr><td> anti-humano CD56-PE/Cy5 </td><td> BECKMAN COULTER </td><td> A07789 </td><td></td></tr><tr><td> anti-humano TCRαβ-PE </td><td> BECKMAN COULTER </td><td> A39499 </td><td></td></tr><tr><td> anti-humano TCR Vγ9-FITC </td><td> BECKMAN COULTER </td><td> IM1463 </td><td></td></tr><tr><td> anti-humano CD27-PE/Cy5 </td><td> BECKMAN COULTER </td><td> 6607107 </td><td></td></tr><tr><td> anti-humano CD45RA-ECD </td><td> BECKMAN COULTER </td><td> IM2711 </td><td></td></tr><tr><td> anti-CD69 humano-PE </td><td> BD </td><td> 555531 </td><td></td></tr><tr><td> anti-humano NKG2D-PE </td><td> BECKMAN COULTER </td><td> A08934 </td><td></td></tr><tr><td> Anti-humal IFNγ-PE </td><td> BECKMAN COULTER </td><td> IM2717U </td><td></td></tr><tr><td> Isotipo IgG1-PE controle </td><td> BECKMAN COULTER </td><td> A07796 </td><td></td></tr><tr><td> Isotipo IgG1 controle-ECD ou PE/Cy5 </td><td> BECKMAN COULTER </td><td> A07797 A07798 </td><td></td></tr></tbody></table>

expansion of human peripheral blood  &gamma;&delta; t cells using zoledronate

Células T γδ podem reconhecer e responder a uma grande variedade de estresse induzido por antígenos, desenvolvendo assim uma atividade ampla inata anti-tumorais e anti-infecciosos. 1 A maioria das células T γδ no sangue periférico tem o Vγ9Vδ2 receptor de células T. Estas células reconhecem o antígeno em um principal de histocompatibilidade forma complexa independente e desenvolver forte citolítica e Th1-like funções efetoras. 1 Portanto, as células T γδ são células efetoras candidato atraente para a imunoterapia do câncer. Vγ9Vδ2 células T responder a phosphoantigens tais como (E)-4-hidroxi-3-metil-but-2-enilo pirofosfato (HMBPP), que é sintetizada em bactérias através de biossíntese de isoprenóides; 2 e isopentenil pirofosfato (IPP), que é produzido em células eucarióticas através da via mevalonato. 3 Na condição fisiológica, a geração de IPP na célula nontransformed não é suficiente para a ativação de células T γδ. Desregulação da via mevalonato em células tumorais leva ao acúmulo de IPP e γδ T ativação das células. 3 Porque aminobisphosphonates (tais como o pamidronato ou zoledronato) inibem sintase farnesil pirofosfato (FPP), a enzima agir a jusante do IPP na via mevalonato, níveis intracelulares de IPP e sensitibity para γδ reconhecimento células T pode ser terapeuticamente aumentou aminobisphosphonates. IPP acumulação é menos eficiente nas células nontransfomred de células tumorais com uma concentração farmacologicamente relevantes da aminobisphosphonates, que nos permitem imunoterapia para o câncer, ativando as células T γδ com aminobisphosphonates. 4 Curiosamente, IPP acumula em monócitos quando PBMC são tratados com aminobisphosphonates, por causa do eficiente absorção de drogas por essas células. 5 monócitos que se acumulam IPP se tornar células apresentadoras de antígenos e estimular Vγ9Vδ2 células T no sangue periférico. 6 Com base nesses mecanismos, nós desenvolvemos uma técnica de expansão em grande escala de culturas de células T γδ usando zoledronato e interleucina -2 (IL-2). 7 Outros métodos para a expansão de células T γδ utilizar o pirofosfato bromohydrin sintéticos phosphoantigens (BrHPP) 8 ou 2-metil-3-butenyl-1-pirofosfato (2M3B1PP). 9 Todos estes métodos permitem que ex vivo de expansão, resultando em um grande número de células T γδ para uso em imunoterapia adotiva. No entanto, apenas zoledronato é um reagente FDA-approved disponíveis comercialmente. Zoledronato-expandida de células T γδ mostrar CD27 - CD45RA - fenótipo de memória efetoras e sua função pode ser avaliada por IFN-γ ensaio de produção 7.

Watch this Scientific Journal Video about Expansion of Human Peripheral Blood  ÃŽÂ³ÃŽÂ´ T Cells using Zoledronate at JoVE.com

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

Um método para expandir células T γδ a partir de células mononucleares do sangue periférico (PBMC) é descrito. PBMC células derivadas de T γδ são estimuladas e expandida usando zoledronato e interleucina-2 (IL-2). Expansão em grande escala de células T γδ podem ser aplicadas a imunoterapia celular autóloga de câncer.

Expansão do sangue periférico humano γδ células T usando zoledronato

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

expansion-of-human-peripheral-blood-t-cells-using-zoledronate

Research

JoVE Journal

Immunology and Infection

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

26.5K visualizações.  University of Tokyo Hospital. Um método para expandir células T γδ a partir de células mononucleares do sangue periférico (PBMC) é descrito. PBMC células derivadas de T γδ são estimuladas e expandida usando zoledronato e interleucina-2 (IL-2). Expansão em grande escala de células T γδ podem ser aplicadas a imunoterapia celular autóloga de câncer.

Vídeo: Expansion of Human Peripheral Blood  γδ T Cells using Zoledronate

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Expansão, Purificação e Avaliação Funcional de Células NK do sangue periférico

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

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 ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Aplicação Clínica de<em&gt; A Bela Adormecida</em&gt; Artificial e Antigen Presenting Cells para modificar geneticamente células T de Sangue do Cordão Umbilical e periférica

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Uma<em&gt; In vitro</em&gt; Modelo para estudar respostas imunes de sangue periférico humano células mononucleares para Vírus Respiratório Sincicial Humano Infecção

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Avaliando a inata Sensing de HIV-1 CD4 Infected<sup&gt; +</sup&gt; As células T por células dendríticas plasmocitóides Usando um<em&gt; Ex vivo</em&gt; Co-cultura do sistema.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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 ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Uma abordagem automatizada-Semi Preparando Antibody Cocktails para Análise imunofenotípica de Sangue Periférico Humano

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

Isolamento e Citometria de Fluxo Analysis of Human Endocervical Gama Cells Delta T

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran aumenta a eficiência de Lentivirus transdução das células NK primário murino e humano

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Expansão do sangue periférico humano γδ células T usando zoledronato

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