Nos gustaría dar las gracias a Aaron Selya útil para el debate. Este trabajo fue apoyado por el NIH subvención AI29575, CA108835, AI077097 a JS, una subvención piloto de la Johns Hopkins malaria Instituto de Investigación y el Departamento de Defensa de conceder PC 040972 para MO

1. Decisiones, HLA-A2-Ig base AAPC <ol><li> Prepare el buffer de borato estéril <ol><li> Disolver el ácido bórico en agua para 0,1 M. Ajustar el pH a 7.0. </li><li> Filtrar a través de un filtro de 0.22μm estéril y se almacenan a 4 ° C. </li></ol></li><li> Prepare el buffer de lavado estéril bolas <ol><li> Tomar 956ml PBS w / o magnesio o calcio. </li><li> Añadir 30 ml de suero humano AB. </li><li> Añadir EDTA 2 mM de concentración final. </li><li> Añadir 0.1gsodium azida. </li><li> Filtrar a través de un filtro de 0,22 micras estéril y se almacenan a 4 ° C. </li></ol></li><li> Tomar 1 ml de perlas de Invitrogen Epoxy M-450 en stock, aproximadamente 400 millones de cuentas (cuentas se pueden contar por hemocitómetro), y poner en un frasco estéril, tornillo de la tapa de cristal. </li><li> Ponga el frasco en contra de un imán Dynal MPC-1, mientras que las cuentas se adhieren a la pared del vial, separar el sobrenadante por aspiración. Lávese las cuentas de una vez con 1 ml de solución tampón de borato. </li><li> Volver a suspender las bolas en una mezcla de 1 ml de solución tampón de borato y 20 mg de HLA-A2-Ig dímero y 20 mg de CD28 mAb anti-humano (clon 9.3). </li><li> Proteína de la conjugación de bolas: poner el frasco de vidrio en los rotadores y girar a 4 ° C durante 24 horas. </li><li> Colocar el tubo en MPC-1 imán y eliminar todos los tampón borato. </li><li> Lave las cuentas dos veces con buffer de lavado de 1 ml de bolas. </li><li> Incubar las cuentas en un tampón de lavado 1 ml de bolas, gire a 4 ° C durante 24 horas. Debido a tampón de lavado bolas contiene suero humano AB, que bloqueará todos los sitios de unión a proteínas residuales en las cuentas. </li><li> Eliminar el sobrenadante del frasco de vidrio, sustituya con 1 ml de solución tampón fresca Lavar bolas. </li></ol> 2. Control de calidad de AAPC y el péptido de carga, almacenamiento <ol><li> Añadir ~ 5 × 10 5 gotas de tampón de lavado FACS 100μl en tubos de FACS, y se tiñen con 1μl de anti-ratón IgG1-PE (reconocer la porción Fc de HLA-A2-Ig) y 1μl de anti-ratón IgG2a-FITC (reconocer la parte Fc de mAb anti-CD28). Después de la tinción durante 20 minutos en FACS buffer de lavado, lavado de nuevo y leer el resultado de la tinción de inmediato por citómetro de flujo (Figura 4). </li><li> Carga de péptidos en las cuentas: lavar las cuentas dos veces en frasco de vidrio con 1 ml de PBS estéril. Resuspender con 1 ml de PBS estéril a continuación, agregar 10μl de CMV péptido (1mg/ml) </li><li> Contar las gotas de hemocitómetro, y la etiqueta del vial con la fecha y la concentración. </li><li> AAPC están listos para su uso después de por lo menos 3 días de incubación del péptido a 4 ° C, para permitir suficiente tiempo de unión del péptido en el dímero HLA-A2-Ig. Las cuentas se pueden almacenar a 4 ° C y seguir funcionando por lo menos durante 6 meses. </li></ol> 3. Aislamiento humano CTL <ol><li> Recoger ~ 100 ml de sangre periférica fresca de salud de los donantes HLA-A2 positivo en 10 tubos de sodio BD Vacutainer de heparina. Use una aguja de calibre 21 o mayor para evitar la hemólisis. </li><li> Centrifugar a 300xg durante 10 minutos a temperatura ambiente </li><li> Retire con cuidado la capa de plasma superior por aspiración. El plasma puede ser utilizado como suplemento para el medio de cultivo. </li><li> Vuelva a colocar el plasma recogido con PBS estéril y la transferencia de la sangre en un frasco de cultivo estéril T75 o 50 ml tubos cónicos. Mezclar la sangre con PBS y pipeteando arriba y hacia abajo. </li><li> Una vez que toda la sangre se recoge, prepara otros cuatro tubos cónicos de 50 ml y agregar 15 ml de Ficoll-Paque Plus. </li><li> Poco a poco superposición 30-35 ml de células de la sangre en la parte superior de la Ficoll de cada tubo. Mantener la interfaz entre los distintos Ficoll y las células sanguíneas. </li><li> Centrifugar a 500xg durante 20 minutos a temperatura ambiente. A su vez el freno de &quot;off&quot; y la aceleración lo más bajo posible para mantener una relación clara entre las capas. </li><li> Con una pipeta seriological, cuidadosamente aspirar la capa de PBMC y recoger las PBMC en un nuevo tubo cónico de 50 ml. Cuando todos los PBMC se cosechan agregar 30 ml de PBS y se giran a 400xg durante 10 minutos. Descartar el sobrenadante y lavar una vez más con 30 ml de PBS para eliminar todos los Ficoll residual. </li><li> Proceder a CD8 + aislamiento de células T mediante el uso de Miltenyi humana de células T CD8 + kit de aislamiento de acuerdo con el protocolo del fabricante. Este kit muy enriquece de las células CD8 + (normalmente&gt; 95%) por el agotamiento de CD8 - las células. </li><li> Cuente los linfocitos T CD8 +. La pureza de la esperada debe ser mayor al 95%. Para confirmar este uso de 2x10 5 células y realizar un análisis CD4/3/8 FACS. El CTL restante puede ser utilizado de inmediato para el antígeno específico de la estimulación AAPC o pueden ser congelados para su uso futuro. </li></ol> 4. En el sistema de cultivo in vitro basado en AAPC <ol><li> Preparar medio de cultivo <ol><li> De TF (de células T del factor de crecimiento, realizado en el laboratorio 4) 2X medio de cultivo: medio RPMI completo, más el 5% de los donantes autólogos de plasma y un 8% el factor de células T de crecimiento. </li><liPlasma&gt; donantes autólogos pueden ser sustituidos por inactivado por calor suero humano AB. </li></ol></li><li> Resuspender 1.000.000 CTL en 8 ml de medio TF cultura 2X más de 8 ml de medio RPMI completo, agregue 1 × 10 6 AAPC, mezclar bien. </li><li> Utilice una pipeta de canales múltiples de células de la placa en 96 y U fondo placas de cultivo de tejidos. (160 l por pocillo) </li><li> Las células de cultivo a 37 ° C, 5% de CO 2 incubater durante 7 días. Alimentar a las células en el día 4 con 80 l / pocillo mediano TF 2X. </li><li> Las células están listas para ser cosechadas en el día 7. Después de la cosecha, el lugar a las células contra el imán y eliminar la AAPC de edad. </li><li> Antígeno específico puede ser determinado por tinción tetrámero de acuerdo con el protocolo del fabricante. Tinción fenotipo y tinción intracelular de citoquinas se realizó de acuerdo a nuestros tres estudios anteriores. </li><li> Las células pueden ser cosechadas replated con AAPC de nuevo en las mismas condiciones. El número de células y la especificidad del antígeno se espera que aumente después de la estimulación repetida. </li></ol> 5. Los resultados representativos: Un ejemplo de AAPC después de la conjugación HLA A2-Ig y anti-CD28 se muestra en la Figura 4. Conjugación de proteínas con éxito es evidente un claro desplazamiento de la tinción de anticuerpos correspondientes. Mientras que la frecuencia de linfocitos T citotóxicos específicos de CMV en la sangre periférica es generalmente 0,5-1%, después de una semana de la AAPC mediada por la estimulación, la especificidad puede alcanzar el 55 - 93% (Figura 2 y 3). La expansión de CTL antígeno específico puede ser muy variable entre los diferentes donantes, pero los resultados son reproducibles dentro de un mismo donante. Por extrapolación, la expansión de las células específicas de CMV puede ser miles de veces en comparación con los niveles de precursor directamente ex vivo (datos no mostrados). Tinción intracelular de citoquinas (Figura 3) muestra que estos CTL ampliado son polifuncionales, más que agotado, después de cultivo celular y la proliferación prolongada significativa. <img src="/files/ftp_upload/2801/2801fig1.jpg" alt="Figura 1"/> Figura 1. Diagrama de flujo del Representante AAPC expansión ex vivo de la base CTL humanos para la inmunoterapia adoptiva en pacientes TCPH alogénicos <img src="/files/ftp_upload/2801/2801fig2.jpg" alt="Figura 2"/> Figura 2. Representante tetrámero de la tinción de linfocitos T citotóxicos específicos generados por CMV AAPC después de una semana de la cultura <img src="/files/ftp_upload/2801/2801fig3.jpg" alt="Figura 3"/> Figura 3. Representante resultado tinción intracelular de citoquinas de linfocitos T citotóxicos específicos generados por CMV AAPC (CMV especificidad fue del 61%) <img src="/files/ftp_upload/2801/2801fig4.jpg" alt="Figura 4"/> Figura 4. Representante de la tinción de la M-450 Epoxy cuentas después de la conjugación de proteínas teñidas con anti-ratón IgG1-PE y anti-ratón IgG2-FITC

<ol>
	<li>Walter, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+cellular+immunity+against+cytomegalovirus+in+recipients+of+allogeneic+bone+marrow+by+transfer+of+T-cell+clones+from+the+donor.">Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.</a> N Engl J Med. 333, 1038-1038 (1995).</li><li>Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cell+transfer:+a+clinical+path+to+effective+cancer+immunotherapy.">Adoptive cell transfer: a clinical path to effective cancer immunotherapy.</a> Nat Rev Cancer. 8, 3669-3669 (2008).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+induction+and+expansion+of+antigen-specific+cytotoxic+T+cells+by+HLA-Ig-coated+artificial+antigen-presenting+cells.">Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells.</a> Nat Med. 9, 619-619 (2003).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+antigen-presenting+cells:+artificial+solution+for+real+diseases.">Artificial antigen-presenting cells: artificial solution for real diseases.</a> Trends Mol Med. 11, 412-412 (2005).</li></ol>

El sistema de AAPC que describimos aquí es un sistema eficiente para la expansión ex vivo de linfocitos T citotóxicos humanos contra una variedad de antígenos. Especial cuidado se debe tomar con respecto a la calidad de la conjugación de la proteína y la distribución uniforme de la AAPC y CTL en el cultivo en placa de 96 pocillos. El uso de este enfoque que hemos sido capaces de ampliar CTL por más de 8 semanas, durante el cual hemos ampliado antígeno-específica CTL hasta un millón de veces 4. Ha habido varios sistemas de APC artificial utilizando líneas de células u otras plataformas acelular 5, sin embargo, de acuerdo con los datos publicados cada sistema tiene su perfil único en lo que respecta a la expansión y la especificidad de soporte de aplicaciones diferentes. Importante, ya que la calidad de CTL es tan importante como la cantidad, la polifuncionalidad de los CTL CMV específicos generados por nuestro sistema se espera que confieren superior anti-viral eficiencia.

<table border="1"><tbody><tr><th> Reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> Vacutainer tubo (contiene heparina) </td><td> Becton Dickinson </td><td> 367874 </td></tr><tr><td> Humana de células T CD8 + Kit de aislamiento </td><td> Miltenyi </td><td> 130-094-156 </td></tr><tr><td> Dynabeads M-450 Epoxy </td><td> Invitrogen </td><td> 140,11 </td></tr><tr><td> Dynal MPC-1 Imán </td><td> Invitrogen </td><td> 120-01D </td></tr><tr><td> Ficoll-Paque Plus </td><td> GE Healthcare </td><td> 17-1440-03 </td></tr><tr><td> Medio RPMI 1640 </td><td> Gibco </td><td> 11875 </td></tr><tr><td> HLA-A2-Ig dímero X </td><td> Becton Dickinson </td><td> 551263 </td></tr><tr><td> iTAgMHC tetrámero (HLA-A2-CMV)-PE </td><td> Beckman Coulter </td><td> T20099 </td></tr><tr><td> Falcon claro de 96 pocillos placa de Microtest </td><td> Becton Dickinson </td><td> 353077 </td></tr><tr><td> Rata anti-ratón IgG2a-FITC </td><td> Becton Dickinson </td><td> 553390 </td></tr><tr><td> Cabra anti-ratón IgG1-PE </td><td> Invitrogen </td><td> P21129 </td></tr><tr><td> Suero humano AB </td><td> Atlanta biológicos </td><td> S40110 </td></tr><tr><td> Ratón anti-humano CD8a-FITC </td><td> Sigma-Aldrich </td><td> F0772 </td></tr><tr><td> Ratón anti-humano CD8a-APC </td><td> Becton Dickinson </td><td> 340684 </td></tr><tr><td> Ratón anti-humano IFN-FITC </td><td> Becton Dickinson </td><td> 340449 </td></tr><tr><td> Mouse anti-TNFa humano-PE </td><td> Becton Dickinson </td><td> 340512 </td></tr></tbody></table>

hla-ig based artificial antigen presenting cells for efficient ex vivo expansion of human ctl

CTL con función efectora óptima desempeñar un papel fundamental en la mediación de la protección contra diversas infecciones intracelulares y el cáncer. Sin embargo, los individuos pueden presentar microambiente inmunosupresores y, en contraste con la activación de CTL, sus células antígeno autólogo presenta tienden a tolerize o anergize CTL antígeno específico. Como resultado de ello, aunque todavía en fase experimental, CTL basado en inmunoterapia adoptiva ha evolucionado hasta convertirse en un prometedor tratamiento para diversas enfermedades como el cáncer y las infecciones de virus. En los experimentos iniciales ex vivo ampliado CMV (citomegalovirus) CTL específicas se han utilizado para el tratamiento de la infección por CMV en pacientes inmunocomprometidos trasplante alógeno de médula ósea. Si bien es común que amenaza la vida viremia por CMV en estos pacientes, ninguno de los pacientes que reciben ampliado CTL desarrollar enfermedades relacionadas con el CMV, lo que implica la inmunidad anti-CMV es establecido por la adoptively transferido CTL 1. Prometedores resultados también se han observado para el melanoma y puede extenderse a otros tipos de cáncer 2. Si bien hay muchas maneras de ex vivo de estimular y ampliar CTL humanos, los enfoques actuales están restringidas por las limitaciones técnicas y de costos. Por ejemplo, el estándar de oro actual se basa en el uso de la DC autólogas. Esto requiere que cada paciente para donar un número importante de los leucocitos y también es muy caro y laborioso. Por otra parte, se detalla en la caracterización in vitro de la DC amplió CTL ha revelado que estos tienen una función efectora sólo subóptima 3. Aquí presentamos un muy eficiente sistema de AAPC base para la expansión ex vivo de los derechos humanos CMV CTL específicas para inmunoterapia adoptiva (Figura 1). La AAPC se hicieron mediante el acoplamiento magnético de células cuentas de tamaño humano con HLA-A2-Ig anti-dímero y CD28mAb 4. Una vez que se hacen AAPC, que puede ser cargado con varios péptidos de interés, y seguir funcionando durante meses. En este informe, AAPC se cargaron con un péptido dominante de CMV, pp65 (NLVPMVATV). Después del cultivo humano purificado CD8 + CTL de un donante sano con AAPC por una semana, el CMV específica CTL se puede aumentar de manera espectacular en la especificidad de hasta el 98% (Figura 2) y se amplifica más de 10.000 veces. Si hay más CMV-CTL específicos se requiere, una mayor expansión se puede conseguir fácilmente por la estimulación repetitiva con AAPC. Caracterización fenotípica y funcional muestra que estas células se han ampliado el fenotipo de un efectoras de memoria y hacer que cantidades significativas de TNF-alfa y IFN (Figura 3).

Watch this Scientific Journal Video about HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL at JoVE.com

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Un nuevo método de CC independiente para la inducción y la expansión de células T específicas de antígeno se describe. HLA A2-Ig células antígeno artificial basada en la presentación (AAPC) se cargan con el HLA-A2 péptidos restringidos a expandirse de manera eficiente CTL del antígeno específico diverso. Esta tecnología tiene un gran potencial para el CTL basado inmunoterapia adoptiva.

HLA-Ig celdas en función de antígeno artificiales presentación para mejorar la eficiencia Ex vivo Ampliación de los Derechos Humanos CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

hla-ig-based-artificial-antigen-presenting-cells-for-efficient-ex

Research

JoVE Journal

Immunology and Infection

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

15.3K vistas.  Johns Hopkins University. Un nuevo método de CC independiente para la inducción y la expansión de células T específicas de antígeno se describe. HLA A2-Ig células antígeno artificial basada en la presentación (AAPC) se cargan con el HLA-A2 péptidos restringidos a expandirse de manera eficiente CTL del antígeno específico diverso. Esta tecnología tiene un gran potencial para el CTL basado inmunoterapia adoptiva.

Video: HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Antígeno Específico de la En Vivo Matar ensayo utilizando CFSE células diana marcadas

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Investigación

Inmunología e Infección

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Expansión ex vivo de células T tumor-reactiva por medio de la 1/Ionomycin briostatina y la formulación de la cadena gamma común de citoquinas

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

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 ...

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

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

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

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

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 ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Antígeno Artificial célula presentadora (AAPC) La activación mediada y expansión de linfocitos T citolíticos naturales

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

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 ...

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 ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Killer Artificial células presentadoras de antígenos (KaAPC) para la utilización eficiente<em&gt; In Vitro</em&gt; El agotamiento de células T humanas específicas de antígeno

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

El aislamiento de linfocitos de la piel humana para el Análisis y fenotípica<em&gt; Ex Vivo</em&gt; Cultivo Celular

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Desarrollo de un modelo de colitis impulsado por el antígeno para el Estudio de la presentación de antígenos por células presentadoras de antígenos a las células T

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...