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  Î³Î´ T Cells using Zoledronate at JoVE.com

Expansion of Human Peripheral Blood  &amp;#947;&amp;#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  &#947;&#948; 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

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 ...

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 ...

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.

Imagem altamente multiplexada e de super resolução de células T usando madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

Avaliação da Interface sináptica de células T humano primário do sangue periférico e tecido linfoide

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

Isolamento, ativação e expansão de células T baseadas em flotação de amostras de células mononucleares do sangue periférico humano usando microbolhas

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14&#8722; mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

Diferenciação de Osteoclastos Funcionais de Monócitos CD14+ do Sangue Periférico Humano

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...

Coleta e crioarmazenamento de PBMC para análise bioenergética mitocondrial

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

Autor em destaque: Preservação de parâmetros bioenergéticos em células mononucleares do sangue periférico após criopreservação 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

Criopreservação e Avaliação Bioenergética de Células Mononucleares do Sangue Periférico Humano

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...