1. Aislamiento de PBMC <ol><li> Extracción de sangre (7,5-8,0 ml) en un tubo BD Vacutainer CPT preparación celular con heparina sódica. El tubo contiene un anticoagulante con heparina sódica y un fluido de densidad de Ficoll-Hypaque, además de una barrera de gel de poliéster, que separa los dos líquidos. Tubo de centrífuga / muestra de sangre a temperatura ambiente (18 ° C a 25 ° C) en un rotor horizontal (se gira hacia fuera de la cabeza) durante 20 minutos a 1800 x g. Interruptor de centrífuga de los frenos. </li><li> Después de la centrifugación, la secuencia de capas se produce de la siguiente manera (visto desde arriba a abajo): un plasma) - b) las células mononucleares de sangre periférica (CMSP) y plaquetas - c) la densidad de la solución - d) de gel de poliéster - e) granulocitos - f) glóbulos rojos (Fig. 1). </li><li> Recoger una fracción de la capa de plasma, dejando 5 a 10 mm de plasma por encima de la interfase sin alterar la capa de células. El plasma puede ser utilizado para el cultivo (sección 2.5). </li><li> Cosecha de la fracción enriquecida (CMSP) en la interfase con una pipeta y transferir a un tubo cónico de 15 ml. </li><li> Lave la PBMC con 10 ml de tampón fosfato salino (PBS), invirtiendo el tubo cinco veces, y centrifugar durante 5 minutos a 400 x g. </li><li> Repita los pasos de lavado dos veces, y luego volver a suspender el pellet de células en 5 ml de PBS. Determinar el número de células. Por lo general, 1,3 x 10 6 células se recuperan de 1 ml de sangre total. </li><li> Determinar la frecuencia y el fenotipo de las células T γδ en PBMC por citometría de flujo (sección 3) (Fig. 2). </li></ol> 2. La expansión de las células T γδ <ol><li> Centrifugar las suspensiones de células en 15 ml tubos cónicos durante 5 minutos a 400 x g a temperatura ambiente, y desechar el sobrenadante. </li><li> Preparar medio de cultivo (CM) mediante la adición de la IL-2 (IL-2) y el zoledronato (Zometa) a concentraciones finales de 1000 UI / ml y M 5, respectivamente. ALyS203 (celular y Ciencia Instituto de Tecnología) o Optmizer (Invitrogen) apoyar la expansión de los medios de comunicación bien de las células T γδ (más información en las referencias 6 y 9). Zometa se suministra en forma líquida (4 mg/5-ml vial). Para preparar una solución de 5 M, se añaden 50 l de Zometa para 30 ml de medio de cultivo. </li><li> Resuspender el sedimento celular en medio de cultivo y se ajustan a 1x10 6 células / ml. </li><li> Pipeta de 1 ml de CM contiene 1x10 6 células en cada pocillo de una placa de 24 pocillos. Para las grandes culturas, las células pueden ser sembradas a 0.5 x 10 6 células / cm 2, de acuerdo a las superficies de los pozos de la placa, plato o recipiente. </li><li> Añadir plasma autólogo (sección 1.3), agrupados suero humano AB, o FCS por lo que es aproximadamente el 10% del volumen de la cultura (100 l por cada pocillo de una placa de 24 pocillos). Coloque las placas en un humidificado 37 ° C, 5% de CO 2 incubadora durante 24-48 horas. </li><li> Mantener el cultivo a una densidad celular de 0,5 a 2 x 10 6 células / ml. Añadir un nuevo soporte de la IL-2 (1000 UI / ml) solamente (sin Zometa) cada 2-3 días, y la transferencia de células cultivadas en pozos nuevos o frascos como sea necesario, de acuerdo con el grado de proliferación celular (Fig. 3). Suministro de plasma o suero al medio de manera que la concentración sérica se puede mantener por lo menos 1%. </li><li> Las células de la cosecha en el día 12-14 y determinar la frecuencia, fenotipo, y las funciones de las células T γδ por citometría de flujo (ver más abajo). </li></ol> 3. El análisis fenotípico por citometría de flujo <ol><li> Transferencia de 200 muestras de l que contiene 2 x 10 5 células de clasificación de células activadas por fluorescencia (FACS) tubos. </li><li> Añadir 2 ml de PBS frío y centrifugar durante 5 minutos a 400 x g. A continuación, volver a suspender las pastillas en 50 l de tampón FACS (PBS + 1% FCS + 0,1% de azida de sodio). Añadir 5 l de cada una de las muestras de anticuerpos (los anticuerpos monoclonales se listan en la Tabla 1). </li><li> Incubar en hielo en la oscuridad durante 20 min. </li><li> Añadir 2 ml de tampón FACS para cada muestra, y luego vórtice. Centrifugue las muestras durante 5 minutos a 400 x g a 4 ° C. Decantar cuidadosamente el sobrenadante. </li><li> Resuspender las células en 300 l de tampón FACS y agitar. Analizar las muestras en un citómetro de flujo (Fig. 2). </li></ol> 4. IFN-γ producción de ensayo de 10 <ol><li> El día antes del ensayo, preparar las células estimulador mediante el cultivo de 3.5 x 10 5 células Daudi / ml en medio RPMI 1640 más el 10% de FCS (RPMI-10) durante la noche con Zometa (5 M) (en adelante designado Z-Daudi). </li><li> Recoger Z-Daudi y resuspender en medio RPMI-10 en 2x10 6 células / ml. Añadir 100 ml de Z-Daudi (2x10 5) a cada pocillo de una placa de fondo redondo de 96 pocillos. </li><li> Prepare las células T γδ en 2x10 6 células / ml en RPMI-10 contiene Brefeldina A a 20 mg / ml. Transferencia de 100 l de suspensión de células T γδ (2x10 5) a cada pocillo que contiene Z-Daudi células o de control de los pozos (100 l de RPMI-10 solamente, o RPMI-10 con 20 ng / ml de forbol 12-miristato 13-acetato[PMA] y 2 mg / ml de ionomicina). </li><li> Mezclar pipeteando arriba y abajo varias veces. Se incuba durante 4 horas en un 37 ° C, 5% de CO 2 incubadora. </li><li> Centrifugar la placa durante 5 minutos a 400 x g a 4 ° C y volver a suspender las pastillas en 200 l de PBS frío. </li><li> Transferencia de las muestras a los tubos de FACS. Agregar 4 ml de PBS frío y centrifugar los tubos durante 5 minutos a 400 x g a 4 ° C. </li><li> Resuspender los pellets en 50 l de tampón FACS con FITC-conjugado anti-TCRVγ9 (5 l) y PE/Cy5-conjugated anti-CD3 (2,5 l). Incubar protegido de la luz durante 15 minutos a temperatura ambiente. </li><li> Añadir 100 ml de reactivo IntraPrep 1 y se incuba durante 15 min a temperatura ambiente. Agregar 4 ml de PBS a cada tubo y centrifugar durante 5 minutos a 400 x g a temperatura ambiente. </li><li> Eliminar el sobrenadante por aspiración y añadir 100 ml de reactivo 2 IntraPrep. Incubar durante 5 minutos a temperatura ambiente sin agitación. </li><li> Añadir 5 l de PE-conjugadas anti-IFN-γ MAB para el tubo de ensayo. Incubar protegido de la luz durante 15 minutos a temperatura ambiente. </li><li> Agregar 4 ml de PBS a cada tubo y centrifugar durante 5 minutos a 400 x g a temperatura ambiente. Eliminar el sobrenadante por aspiración y resuspender el botón celular en 0,5 ml de tampón FACS. </li><li> Analizar las células por citometría de flujo. Puerta de CD3 + + TCRVγ9 células y examinar la expresión de IFN-γ (Fig. 4). </li></ol> 5. Los resultados representativos: Es importante determinar el porcentaje de células T γδ en PBMC en el inicio de la cultura. Como se muestra en la figura. 2 A, el porcentaje de linfocitos CD3 + TCRVγ9 + células T γδ en PBMC fue del 1,6% en el día 0. Las poblaciones dominantes fueron CD27 + CD45RA + CD27 o ingenuo CD45RA + - fenotipos memoria central. Cuando las células T γδ fueron estimulados de manera eficiente, se formaron grupos en los días 3-5 (Fig. 3 A y B). Cuando la formación de agrupaciones se demoró, el ​​crecimiento de otros tipos de células, como células CD4 + y CD8 + αβ células T o células NK podría dominar el crecimiento de las células T γδ (Fig. 3 C y D). Después de 14 días de cultivo, la frecuencia de las células T γδ aumentó a más del 93,8% de las células cultivadas en el éxito de las culturas de las células T γδ (Fig. 2 E). Los cultivos de células T γδ upregulated NKG2D y expresión de CD69 (Fig. 2 G y H). La exhibición de CD27 - CD45RA - fenotipo efectoras de memoria (Fig. 2 F). Las funciones de las células T γδ fueron evaluados con respecto a la producción de citocinas y citotoxicidad. El IFN-γ intracelular tinción demostrado que las células T γδ produjo IFN-γ en respuesta a PMA / ionomicina tratamiento o células Daudi que Z-IPP acumulado después de un tratamiento zoledronato (Fig. 4). Estos resultados indican que el zoledronato eficiente puede estimular y expandir las células T γδ funcional. <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> IgG1 de ratón </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> IgG1 de ratón </td><td> IgG1 de ratón </td></tr></tbody></table> Tabla 1. Los anticuerpos monoclonales utilizados en la tinción multicolor de las células T γδ. Un ejemplo del análisis fenotípico de las células T γδ realizado en nuestro laboratorio se muestra en la figura. 2. <img src="/files/ftp_upload/3182/3182fig1.jpg" alt="Figura 1"/> Figura 1. Separación de PBMC. Sangre (7,5-8,0 ml) se introduce en un tubo BD Vacutainer CPT preparación celular con heparina sódica y directamente se centrifuga durante 20 minutos a 1800 x g. Después de la centrifugación, las capas que resulta como se ve desde arriba a bottom: un Plasma) - b) PBMC y plaquetas - c) Densidad de la solución - d) de gel de poliéster - e) Granulocitos - f) Los glóbulos rojos. <img src="/files/ftp_upload/3182/3182fig2.jpg" alt="Figura 2"/> Figura 2. Fenotipo de superficie típico de las células T γδ. PBMC fueron estimulados con zoledronato e IL-2 durante 14 días. Las células se tiñeron con FITC anti-TCR Vγ9 y PE/Cy5-labeled anti-CD3 para controlar la expansión de las células T γδ (A y E). Las células T γδ fueron identificados por su expresión de TCRVγ9, y la expresión de CD27 y CD45RA (B y F), NKG2D (C y G), o CD69 (D y H) se examinó. <img src="/files/ftp_upload/3182/3182fig3.jpg" alt="Figura 3"/> Figura 3. Representante γδ cultivos de células T. PBMC fueron estimulados con IL-2 (1000 UI / ml) y el zoledronato (5 M). Campos representativos se muestran (IX71 microscopio invertido [Olympus] x 200). Las agrupaciones y los agregados de células T γδ se puede observar en el día 3 (A) y el día 5 (B), cuando las células T γδ se ampliaron con éxito. Por el contrario, existen grupos o agregados se observaron cuando el crecimiento de células T γδ no era la adecuada (C y D). <img src="/files/ftp_upload/3182/3182fig4.jpg" alt="Figura 4"/> Figura 4. IFN-γ producción. γδ células T se incubaron con RPMI-10 sólo (A) o PMA / ionomicina (B) o Z-Daudi (C) durante 4 horas. En primer lugar, la expresión superficial de TCRVγ9 estaba manchada y IFN-γ producción fue examinado por intracelular de IFN-γ tinción. <img src="/files/ftp_upload/3182/3182fig5.jpg" alt="Figura 5"/> Figura 5. Cinética de la cultura de las células T γδ. (A) Número absoluto de células en cultivo, (B) el porcentaje de células T γδ, y (C) número absoluto de células T γδ en los puntos de tiempo indicado.

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No hay conflictos de interés declarado.

El método aquí presentado permite la expansión eficiente de las células T γδ a partir de PBMC. γδ células T activadas y ampliado por zoledronato y la IL-2 desarrollan completar las funciones efectoras, se refleja en la producción de citocinas y citotoxicidad. Se ha informado de que el sintético phosphoantigens pirofosfato bromhidrina (BrHPP) y 2-metil-3-butenil-1-pirofosfato (2M3B1PP) también ampliar las células T γδ, sin embargo, no están disponibles comercialmente. En contraste, el zoledronato es que y...

<table border="1"><tbody><tr><th> Reactivo nombre </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (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 Celular preparación del tubo con 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 medianas </td><td> Celular y Ciencia Instituto de Tecnología </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-humano CD3-FITC o PE/Cy5 </td><td> BECKMAN COULTER </td><td> A07746 FITC A07749 PE/Cy5 </td><td></td></tr><tr><td> anti-CD4 humano-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-IFN-PE humal </td><td> BECKMAN COULTER </td><td> IM2717U </td><td></td></tr><tr><td> Mouse IgG1 control de isotipo-PE </td><td> BECKMAN COULTER </td><td> A07796 </td><td></td></tr><tr><td> Mouse IgG1-ECD control de isotipo o 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

Humanos γδ células T pueden reconocer y responder a una amplia variedad de estrés inducido por antígenos, por lo tanto el desarrollo de una amplia actividad innata anti-tumoral y anti-infecciosos. 1 La mayoría de las células γδ T en sangre periférica tienen el receptor de células T Vγ9Vδ2. Estas células reconocen al antígeno en un complejo mayor de histocompatibilidad-de manera independiente y desarrollar una fuerte funciones efectoras citolítica y como Th1-1. Por lo tanto, las células γδ T son células efectoras candidato atractivo para la inmunoterapia del cáncer. Vγ9Vδ2 células T responden a phosphoantigens ejemplo, (E)-4-hidroxi-3-metil-but-2-enil pirofosfato (HMBPP), que se sintetiza en las bacterias a través de la biosíntesis de isoprenoides, 2 y isopentenil pirofosfato (IPP), que se produce en las células eucariotas a través de la vía del mevalonato. 3 En condiciones fisiológicas, la generación de IPP en células no transformadas no es suficiente para la activación de células T γδ. La desregulación de la vía del mevalonato en las células tumorales conduce a la acumulación de la IPP y la activación de las células T γδ. 3 Porque aminobisfosfonatos (como pamidronato o zoledronato) inhiben la farnesil pirofosfato sintetasa (FPPS), la enzima actúa aguas abajo de la IPP en la vía del mevalonato, los niveles intracelulares de IPP y sensitibity a γδ T reconocimiento de las células pueden ser terapéuticamente aumentó aminobisfosfonatos. IPP acumulación es menos eficiente en las células nontransfomred que las células tumorales con una concentración farmacológicamente relevantes de aminobisfosfonatos, que nos permiten la inmunoterapia para el cáncer mediante la activación de las células T γδ con aminobisfosfonatos. 4 Curiosamente, IPP se acumula en los monocitos al PBMC se tratan con aminobisfosfonatos, debido a la eficiente absorción del fármaco por estas células. 5 monocitos que se acumulan IPP ser células presentadoras de antígeno y estimular Vγ9Vδ2 las células T en la sangre periférica. 6 Basado en estos mecanismos, hemos desarrollado una técnica para la expansión a gran escala de cultivos de células T γδ con zoledronato y la interleucina -2 (IL-2). 7 Otros métodos para la expansión de las células T γδ utilizar el sintético de pirofosfato phosphoantigens bromhidrina (BrHPP) 8 o 2-metil-3-butenil-1-pirofosfato (2M3B1PP). 9 Todos estos métodos permiten ex la expansión in vivo, lo que resulta en un gran número de células T γδ para su uso en inmunoterapia adoptiva. Sin embargo, sólo el zoledronato es un reactivo aprobado por la FDA disponibles en el mercado. Zoledronato-ampliado las células T γδ pantalla CD27 - CD45RA - fenotipo efectoras de memoria y su función puede ser evaluado por el IFN-γ ensayo de producción de 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  &#947;&#948; T Cells using Zoledronate

Un método para expandir las células T γδ a partir de células mononucleares de sangre periférica (CMSP) se describe. PBMC derivados de las células T γδ se estimulan y se expandió con zoledronato y la interleucina 2 (IL-2). La expansión a gran escala de las células T γδ se puede aplicar a la inmunoterapia celular autólogo de cáncer.

Expansión de la sangre periférica humana de células T γδ con 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

26.6K vistas.  University of Tokyo Hospital. Un método para expandir las células T γδ a partir de células mononucleares de sangre periférica (CMSP) se describe. PBMC derivados de las células T γδ se estimulan y se expandió con zoledronato y la interleucina 2 (IL-2). La expansión a gran escala de las células T γδ se puede aplicar a la inmunoterapia celular autólogo de cáncer.

Video: 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.

Expansión, purificación y evaluación funcional de la sangre periférica humana células NK

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

Investigación

Inmunología e Infección

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.

La generación de células T reguladoras inducidas de células humanas primarias T vírgenes y de memoria

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.

Aplicación clínica de<em&gt; La Bella Durmiente</em&gt; Artificial y células presentadoras de antígenos para modificar genéticamente las células T de la sangre del cordón umbilical y 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.

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.

La evaluación de la innata de detección de VIH-1 CD4 infectados<sup&gt; +</sup&gt; Células T por las células dendríticas plasmocitoides Usando un<em&gt; Ex vivo</em&gt; Co-cultura del 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.

Generación de células T humanas aloantígeno-específicos de la sangre periférica

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.

Un enfoque semi-automatizado de preparación de cócteles de anticuerpos para el Análisis Inmunofenotípicos de la sangre periférica humana

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.

Aislamiento y Análisis de citometría de flujo, de 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 ...

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.

Dextrano mejora la eficiencia de transducción lentivirales de las células NK primaria murinas y humanas

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.

Expansión de la sangre periférica humana de células T γδ con zoledronato

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Capítulos en este video

Expansión, purificación y evaluación funcional de la sangre periférica humana células NK

La generación de células T reguladoras inducidas de células humanas primarias T vírgenes y de memoria

Aplicación clínica de

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Dextrano mejora la eficiencia de transducción lentivirales de las células NK primaria murinas y humanas

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