Me gustaría agradecer a Bianca Mothé, Oseroff Carla y Marie-France DelGuercio por haberme introducido en ensayos inmunológicos y el manejo del ratón. Trabajar en el laboratorio de GA Splitter es financiado por el NIH subvención 1-SR1-AI-073558, GLRCE conceder 1-U54-AI-057153, y BARD Estados Unidos-3829-06.

1. Provocando las metas específicas de Efectos CTL mediante la inmunización (día 0) <ol><li> Antes de empezar, decidir sobre un efector específicos: sistema de destino. En este ejemplo de un clásico ensayo in vivo del CTL, vamos a utilizar una vacuna de péptido purificado específico para obtener células T CD8 +. El mismo péptido se utilizará para el pulso de las células diana singénicas, la creación de un efector específicos: sistema de destino. Si lo prefiere, puede optar por utilizar patógeno total o proteínas para obtener su efectores y objetivos específicos. </li><li> Descontaminar la superficie de trabajo en el interior de una campana de bioseguridad para garantizar la esterilidad de las inyecciones. Preparar la vacuna de péptido, en primer lugar la colocación de 50 mg de péptido purificado en 100 l de DMSO al 10% en PBS / ratón en un tubo de 2 ml. A continuación, agregue una cantidad igual de frío adyuvante incompleto de Freund y hasta la marca de 0,2 ml con 1 mm para la mezcla de granos. Desde este punto, mantener inmunógeno en hielo hasta la inyección. Asegure la tapa con Parafilm. </li><li> Use un batidor de cuentas establecido en RPM máximo para mezclar la emulsión durante 3 minutos, inmediatamente la colocación de la emulsión de vuelta en el hielo después de la mezcla. La emulsión resultante debe ser similar en consistencia a la mayonesa. Permita que la emulsión que se siente a 4 ° C durante la noche si el tiempo lo permite (hasta una semana), la mezcla aún más si la separación se nota. </li><li> Use una jeringa de 1 ml y 20 o 20,5 aguja para levantar la emulsión, teniendo cuidado de no tirar las bolas (cuentas debe ser mayor que calibre de la aguja, pero tenga en cuenta). </li><li> Ratón seguro para la inyección subcutánea en la base de la cola. Nosotros usamos un tubo de plástico de 50 ml modificado cónica de manera segura y suave inmovilizar el ratón para inyección. Inyectar el ratón con 0,2 ml de emulsión. </li></ol> 2. Recolección de las células diana (Día 8) <ol><li> Usted tendrá que ingenuo ratones singénicos de las células de su objetivo. La eutanasia a los ratones ingenuo de acuerdo con las normas AAALAC. </li><li> Desinfectar el ratón por aspersión 70% de etanol para reducir al mínimo la posibilidad de contaminación del pelo del ratón u otros contaminantes externos. </li><li> Usando tijeras de disección que cortar pequeños a través de la piel en el lado izquierdo del ratón. Tire de colgajo de piel para descubrir más de saco peritoneal y órganos internos. </li><li> Cortar a través del peritoneo, teniendo cuidado de no comprometer ninguno de los órganos subyacentes. El bazo se encuentra cerca de la superficie y es similar en color al hígado (rojo oscuro), pero más pequeño y más frijol en forma. Los ratones infectados en general, muestran el bazo notablemente mayor que los controles no infectados. </li><li> Retire con cuidado el bazo, teniendo cuidado de ser suave, para no romper el bazo en varias piezas con el fin de recuperar el número máximo de células. Colocar inmediatamente el bazo en una cónica de 15 ml que contienen 10 ml de medio preparado en el hielo. Continuar con los ratones restantes. </li><li> Vierta el contenido del tubo cónico de 15 ml (los medios de comunicación y el bazo) en un triturador de tejidos de vidrio. Triturar el bazo con un montón de fuerza en el brazo superior. Cuando todo el tejido ha perdido su color se puede suponer que se ha recuperado todas las células del tejido conectivo. </li><li> Vierta el contenido del molino a través de un filtro de 70 micras de células en un tubo cónico de 50 ml. Enjuague molino con medio fresco y se vierte a través de un colador de la célula. </li><li> Se centrifuga a 4 ° C, 1300 rpm durante 7 minutos. Elimínese el líquido sobrenadante. </li><li> Resuspender en 10 ml de reactivo de lisis preparado e incubar a 37 º durante 7 minutos. </li><li> Agregue hasta 50 ml con medio fresco y centrifugar, igual que el anterior. </li><li> Resuspender en 50 mL medios de comunicación fresca y centrifugar, igual que el anterior (paso de lavado). * Nota: En este momento se puede realizar una separación de células magnético para un tipo específico de célula (es decir, los macrófagos), si una población de células diana puro se desea para su transferencia a los ratones receptores. Además, en este momento usted podría introducir un tratamiento experimental en la cultura con el fin de estudiar los efectos de que el tratamiento de matar a células específicas. </li><li> Recuento de células y la placa de 1 x 10 7 por pocillo en 100μL de una placa de 96 cultivos de fondo y todo el tejido tratado. * Esta es una alta concentración de células / bien, pero debido a que el ensayo es una labor más rápida, no hemos visto la pérdida de la viabilidad de las células en esta concentración. </li></ol> 3. Tratamiento y etiquetado de las células diana <ol><li> Células pulso adecuado y se replica con el péptido patógeno específico o de control de péptido irrelevante: Añadir 1 mg / ml de péptido. Incubar a 4 ° C durante 1,5 horas. Mover a 37 ° C incubadora para los últimos 30 minutos. Durante este tiempo, preparar reactivos CFSE. </li><li> Prepare CFSE de acuerdo con las especificaciones del fabricante y se diluye a una concentración de trabajo entre 0,5 y 25 micras. Hacer &quot;bajo&quot; la concentración 10 veces menor que la CFSE &quot;alto&quot; de concentración. Por ejemplo, para el [bajo] añadir 0,5 l CFSE a 10 ml de PBS y de [alto], añadir 5 CFSE l de 10 ml de PBS. CFSE calientes preparadas a 37 ° C antes de su etiquetado. </li><li> Retire la placa de las células de la incubadora y el pEllet en una centrífuga de alta velocidad a temperatura ambiente, 1300 rpm durante 7 minutos. Flick para eliminar el sobrenadante. </li><li> Resuspender las células en cualquiera de [baja] o [alto] CFSE añadiendo 200 l de reactivo apropiado para los pozos correspondientes y se incuba durante 15 minutos a 37 ° C. </li><li> Retire la placa de las células de la incubadora y el pellet de nuevo a temperatura ambiente, 1300 rpm durante 7 minutos. Flick para eliminar el sobrenadante. </li><li> Resuspender las células en 200 l / pocillo de medio fresco calentado y se incuba durante 30 minutos a 37 ° C. </li><li> Retire la placa de las células de la incubadora y el pellet a temperatura ambiente, 1300 rpm durante 7 minutos. Flick para eliminar el sobrenadante. </li><li> Resuspender las células en 100 l / pocillo de PBS. </li><li> Para la transferencia adoptiva, añadir un bien [bajo] las células marcadas con un bien [alto] etiquetados células por ratón. Resultante de la inyección debe ser de 50% los objetivos específicos y 50% de células en 0,2 mL irrelevante. </li><li> No te olvides de dejar de lado algunas células marcadas para actuar como su control no transferidos durante el análisis de datos y para el funcionamiento del citómetro de flujo (ver Figura 1). </li></ol> 4. La transferencia adoptiva <ol><li> Anestesiar a los ratones inmunizados previamente péptido (Del part # 1 del presente Protocolo), de acuerdo a las normas de AAALAC. </li><li> Inyectar 0,2 mL de las células diana a través de la etiqueta retro-orbital ruta en los ratones receptores. </li><li> Esperar 6 horas o tiempo predeterminado óptima antes de la eutanasia a los ratones receptores. </li></ol> 5. La cosecha células marcadas * Nota: Los pasos 5.1 a 5.11 son los mismos que los pasos 2.1 a 2.11 <ol start="12"><li> Recuento de células y la placa a la 1 x 10 6 por pozo en 100 L de 96 cultivos de fondo y el tejido alrededor de la placa tratada. </li><li> Fijar las células recuperadas y no transferidos por la adición de 100 l de paraformaldehído al 2% a una concentración final de 1% de paraformaldehído a todos los pocillos. </li></ol> 6. Citometría de flujo y análisis de datos <ol><li> Corra las muestras en un citómetro de flujo y análisis de los datos. </li></ol> 7. Resultados representante Un pico de la disminución de epítopo específico en el histograma resultante es un indicador de la muerte de las células específicas (Figura 1). Use las siguientes ecuaciones (aplicado en la Tabla 1) para determinar el valor real de la lisis de células específicas: Ratio = Porcentaje irrelevante: Porcentaje epítopo específico (pico [bajo]: [alto] pico), Porcentaje específico de lisis = [1 - (para no transferir el control de relación / proporción Experimental)] x 100 <img src="/files/ftp_upload/2250/2250fig1.jpg" alt="Figura 1" /> Figura 1. CFSE perfil de los números de recuperar BALB / c esplenocitos de ratón. Indican el porcentaje de células del fenotipo CFSE alto o bajo. A) no transfirió el control. Todos los [altos] CFSE picos representan epítopo células diana específicas, mientras que [bajo] CFSE picos representan los objetivos de pulso con el péptido irrelevante. B) Las células CFSE etiquetados recuperado de ratón PBS receptor inmunizado (control). C) Las células CFSE etiqueta se recuperó de dos ratones diferentes péptidos destinatario específico, tenga en cuenta los picos de epítopo específico disminuido. <img src="/files/ftp_upload/2250/2250fig2.jpg" alt="Figura 2" /> Figura 2. Representación gráfica de los datos analizados. Tres puntos de datos se muestra para cada grupo de ratones, PBS, péptido A, y B. Péptidos <table border="1"><tbody><tr><td> Muestra </td><td> Epítopo CFSE% Pulsada de Alto </td><td> CFSE irrelevante% BAJA </td><td> Low Ratio: Alto </td><td> Matar por ciento epítopo específico </td></tr><tr><td> PBS </td><td> 42.8 </td><td> 57.2 </td><td> 1.34 </td><td> 16.97 </td></tr><tr><td> PBS </td><td> 43.1 </td><td> 56.9 </td><td> 1.32 </td><td> 15.94 </td></tr><tr><td> PBS </td><td> 44.3 </td><td> 55.7 </td><td> 1.26 </td><td> 11.74 </td></tr><tr><td> Un péptido </td><td> 39.7 </td><td> 60.3 </td><td> 1.52 </td><td> 32.56 </td></tr><tr><td> Un péptido </td><td> 38.6 </td><td> 61.4 </td><td> 1.59 </td><td> 35.61 </td></tr><tr><td> Un péptido </td><td> 40.6 </td><td> 59.4 </td><td> 1.46 </td><td> 29.99 </td></tr><tr><td> Péptido B </td><td> 38.7 </td><td> 61.3 </td><td> 1.58 </td><td> 24.68 </td></tr><tr><td> Péptido B </td><td> 38.5 </td><td> 61.5 </td><td> 1.60 </td><td> 25.32 </td></tr><tr><td> Péptido B </td><td> 39.3 </td><td> 60.7 </td><td> 1.54 </td><td> 22.76 </td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr><tr><td> PBS NT de control </td><td> 47.4 </td><td> 52.6 </td&gt; <td> 1.11 </td><td></td></tr><tr><td> Pep Un control de NT </td><td> 49.4 </td><td> 50.6 </td><td> 1.02 </td><td></td></tr><tr><td> Pep B NT de control </td><td> 45.6 </td><td> 54.4 </td><td> 1.19 </td><td></td></tr></tbody></table> Tabla 1. El análisis de datos representativos. &quot;NT&quot; no es transferido.

<ol>
	<li>Blattman, J. N., Wherry, E. J., Ha, S. J., van der Most, R. G., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+epitope+escape+on+PD-1+expression+and+CD8+T-cell+exhaustion+during+chronic+infection.">Impact of epitope escape on PD-1 expression and CD8 T-cell exhaustion during chronic infection.</a> J Virol. 83 (9), 4386-4394 (2009).</li><li>Jenkins, M. R., Tsun, A., Stinchcombe, J. C., Griffiths, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+strength+of+T+cell+receptor+signal+controls+the+polarization+of+cytotoxic+machinery+to+the+immunological+synapse.">The strength of T cell receptor signal controls the polarization of cytotoxic machinery to the immunological synapse.</a> Immunity. 31 (4), 621-631 (2009).</li><li>Ingulli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+tolerance+and+immunity+in+vivo+by+CFSE-labeling+of+administered+cells.">Tracing tolerance and immunity in vivo by CFSE-labeling of administered cells.</a> Methods Mol Biol. 380, 365-376 (2007).</li><li>Durward, M. A., Harms, J., Magnani, D. M., Eskra, L., Splitter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discordant+Brucella+melitensis+antigens+yield+cognate+CD8++T+cells+in+vivo.">Discordant Brucella melitensis antigens yield cognate CD8+ T cells in vivo.</a> Infect Immun. 78 (1), 168-176 (2010).</li><li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J Immunol Methods. 171 (1), 131-137 (1994).</li><li>Weston, S. A., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+fluorescent+dyes+for+lymphocyte+migration+studies.+Analysis+by+flow+cytometry+and+fluorescence+microscopy.">New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy.</a> J Immunol Methods. 133 (1), 87-97 (1990).</li><li>Akondy, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yellow+fever+virus+vaccine+induces+a+broad+and+polyfunctional+human+memory+CD8++T+cell+response.">The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8+ T cell response.</a> J Immunol. 183 (12), 7919-7930 (2009).</li><li>Wallace, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+antigen-driven+responses+by+flow+cytometry:+monitoring+proliferation+by+dye+dilution.">Tracking antigen-driven responses by flow cytometry: monitoring proliferation by dye dilution.</a> Cytometry A. 73 (11), 1019-1034 (2008).</li><li>Labadi, A., Balogh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+preferences+in+serosal+homing+and+distribution+of+peritoneal+B-cell+subsets+revealed+by+in+situ+CFSE+labeling.">Differential preferences in serosal homing and distribution of peritoneal B-cell subsets revealed by in situ CFSE labeling.</a> Int Immunol. 21 (9), 1047-1056 (2009).</li><li>Suffner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+support+homeostatic+expansion+of+Foxp3++regulatory+T+cells+in+Foxp3.LuciDTR+mice.">Dendritic cells support homeostatic expansion of Foxp3+ regulatory T cells in Foxp3.LuciDTR mice.</a> J Immunol. 184 (4), 1810-1820 .</li></ol>

No hay conflictos de interés declarado. Los experimentos con animales se realizaron de acuerdo con las directrices y normas establecidas por el Comité Institucional y el empleo Comisión (IACUC) de la Universidad de Wisconsin-Madison.

Este ensayo puede ser modificado para investigar la capacidad proliferativa de las células, incluyendo la expansión clonal, porque CFSE relativamente divide en partes iguales entre la progenie 10,11. También es posible utilizar el etiquetado CFSE para investigar la migración de células 6,12, aunque hay otra célula de seguimiento métodos que pueden ser más apropiados en función de su hipótesis, por ejemplo, tetrámeros y bioluminiscencia 13, que también se puede combinar con el...

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antigen specific in vivo killing assay using cfse labeled target cells

Diacetato de carboxifluoresceína succinimidil ester (CFSE) puede ser utilizada para etiquetar con facilidad y rapidez una población celular de interés para la investigación en vivo. Este etiquetado tradicionalmente se ha utilizado para estudiar la proliferación y migración. En el método que aquí se presenta, hemos acortado la línea de tiempo después de la transferencia adoptiva de mirar la supervivencia y la muerte de las células CFSE epítopo específico etiqueta meta 6.4. El nivel de muerte específicas de un clon de células T CD8 + pueden indicar la calidad de la respuesta, ya que su cantidad puede ser engañosa. Específicas de células T CD8 + pueden llegar a ser funcionalmente agotado el tiempo con una disminución en la producción de citoquinas y matando a 7,8. Además, ciertos CD8 + clones de células T no se puede matar, así como otras con diferentes especificidades TCR 9. De la inmunidad mediada por células efectiva (CMI), los antígenos deben ser identificados que producen no sólo un número adecuado de responder las células T, sino también funcionalmente robusto responder las células T. Aquí se evalúa la eliminación de las células específicas por ciento de los dos péptidos clones específicos de células T en ratones Balb / c.

Watch this Scientific Journal Video about Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells at JoVE.com

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Muchas infecciones provocan una respuesta CTL fuerte, pero de vez en cuando, la cantidad de células de respuesta no se correlaciona con el control del patógeno<sup&gt; 1</sup&gt;. Una medida de la calidad del CTL es su capacidad de matar específicamente<sup&gt; 2</sup&gt;. CFSE etiquetado de las células diana pueden ser utilizados para investigar la calidad de la respuesta CTL<em&gt; In vivo</em<sup&gt; 3,4</sup&gt;.

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.  ...

antigen-specific-in-vivo-killing-assay-using-cfse-labeled-target-cells

Research

JoVE Journal

Immunology and Infection

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

19.5K vistas.  University of Wisconsin-Madison. Muchas infecciones provocan una respuesta CTL fuerte, pero de vez en cuando, la cantidad de células de respuesta no se correlaciona con el control del patógeno 1. Una medida de la calidad del CTL es su capacidad de matar específicamente 2. CFSE etiquetado de las células diana pueden ser utilizados para investigar la calidad de la respuesta CTL In vivo

Video: Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

Trans-vivo hipersensibilidad de tipo retardada Ensayo para Antigen Reglamento Específico

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Investigación

Inmunología e Infección

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 ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Desarrollo de derivados de células madre Las células T reguladoras específicas de antígeno contra la autoinmunidad

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

Detección y enriquecimiento de Rare células B específicas de antígeno para el análisis del fenotipo y la función

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

En Vivo Ensayo para la detección de la función citolítica de células T específicas de antígeno utilizando un modelo de vacunación

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

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

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

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

Generación de anticuerpos monoclonales humanos discriminativo de células B antígeno específicas raras circulan en sangre

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

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

Un análisis de Microarray de proteínas para la determinación serológica de anticuerpos las respuestas siguientes infección por Clostridium difficile específica de antígeno

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Opsonofagocítico asesinato ensayo para evaluar las respuestas inmunológicas contra patógenos bacterianos

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Cuantificación de células CD4+ T específicas de antígenos humanos que utilizan éster de succinidilo de carboxifluoresceína

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

Generación de alta eficiencia de células T citotóxicas de ratón primario específicas de antígenos para pruebas funcionales en un modelo de diabetes autoinmune

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...