Name: rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development
Uploaded: 2018-06-19T13:00:00.000+00:00
Duration: 9 min 3 s
Description: Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

Estamos agradecidos a nuestros asistentes técnicos: Bröder U. y H. Shkarlet, que nos ayudaron durante procedimientos experimentales. Este trabajo fue financiado en parte por convocatoria (UniVax, contrato no. 601738 y TRANSVAC2, contratación Nº 730964) y una donación de la Asociación Helmholtz (HAI-IDR). Las fuentes de financiación no influyó en la investigación de diseño, generación del manuscrito o la decisión de presentar para su publicación.

Todos los ratones utilizados en este estudio fueron el fondo C57BL/6. Todos los animales se mantuvieron en condiciones libres de patógenos. Todos los experimentos fueron realizados según la normativa de la ley de protección animal alemán (TierSchG BGBl. S 1105; 25.05.1998) y fueron aprobados por el Comité de Sajonia inferior sobre la ética de los experimentos animales y la Oficina Estatal de baja Sajonia Estatal Oficina de protección al consumidor y seguridad alimentaria, bajo el permiso número 33.4-42502-04-13/1281 y 162280.
1. CFSE tinción de OT-I T las células y transferencia adoptiva
Nota: OT-los ratones son animales transgenically generados que expresan un receptor de célula T (TCR) con fijo α y β cadenas que reconocen a los péptidos inmuno-dominante de OVA, SIINFEKL10,11. Como resultado, estos ratones tienen un número considerablemente alto de CD8 específicas SIINFEKL+ T las células (97%)12 en comparación con ratones normales o vacunados de OVA (≤ 1%)13.
<ol><li>Aislamiento de trazabilidad CD8+ T células de OT-los ratones que expresaban el linfocito T específico Thy1un (Thy1.1) alelo:<ol>
<li>Eutanasia de OT 6-9 semanas de edad-ratones por la inhalación de CO2 seguido por dislocación cervical14. Disecar el bazo y ganglios linfáticos principales (sólo pares inguinales, axilares y cervicales) cortando la piel con tijeras quirúrgicas, separar la piel del cuerpo con la ayuda de pinzas quirúrgicas y fórceps y colocar en cajas Petri (60 x 15 mm) cada uno con un 100 μm poro malla taza para lanzamiento de pulido y de la célula del tejido.</li>
		<li>Mantener platos de petri (cada uno con malla de una taza) en 3-5 mL de Medio RPMI completo (RPMI 1640, 10% v/v FCS, 100 U/mL de penicilina, estreptomicina 50 μg/mL) en hielo.</li>
		<li>Los órganos con la ayuda de un émbolo o un instrumento estéril similar el puré (corte previo no es necesario) y recoger la suspensión unicelular resultante en tubos de centrífuga de 15 mL.</li>
		<li>Centrifugue la suspensión de células a 300 x g durante 10 min a 4 ° C. Deseche el sobrenadante. Lavar las células en 10 mL de PBS frío mediante centrifugación y lisar los eritrocitos del bazo por volver a suspender el sedimento en 1 bazo mL de tampón de cloruro de amonio (tampón de ACK, comercialmente disponible). Incubar las células durante 1,5 min en hielo y posteriormente lavar las células con 10 mL de PBS frío por centrifugación (como antes).</li>
		<li>Vuelva a suspender el sedimento en el mismo tubo en 1 mL de PBS (pH 7.2) que contiene 5% suero bovino fetal de un aislamiento electromagnético.</li>
	</ol>
</li>
	<li>Para realizar el aislamiento magnético, proceder a la selección negativa de CD8+ T las células utilizando un kit de aislamiento magnético según las instrucciones del fabricante o publicado protocolos15.</li>
	<li>Para realizar la tinción de la CFSE, contar primero el número de CD8+ T las células obtienen de OT-I ratones utilizando un contador de células automatizado (contador de partículas). Mancha 1-5 x 107 células/mL en un volumen de 1 a 5 mL con 5 μm CFSE en PBS durante 7 minutos a 37 ° C, protegido de la luz en un tubo falcon de 15 ml.</li>
	<li>Sacian la CFSE tinción añadiendo el mismo volumen (1:1) de suero fetal bovino a las células e Incube por adicional 7 min a 37 ° C, protegido de la luz. Lavar las células con 10 mL de PBS dos veces.</li>
	<li>Contar las células usando un contador de células automatizado. Establecer el número de células al 3-5 x 107 células/mL de PBS para inyectar por vía intravenosa 3-5 x 106 células/ratón de 100 μl (i.v.) a través de la vena de la cola.</li>
	<li>Para realizar inyecciones de vena de la cola, inmovilizar los ratones en restrainers apropiados. Caliente la zona de la espalda y la cola de los ratones que se inyecta usando una lámpara de luz roja, entre 20 a 25 cm de los ratones por 1-3 min permitir la vasodilatación de la vena de la cola. Nota: Esto ayuda a facilidad vena detección y administración de la suspensión de células.</li>
	<li>Asegurar (con la mano colocada entre la lámpara y los ratones) que no es demasiado caliente para los ratones y cuando las venas de la cola son claramente visibles, proceder a la inyección. Inyectar las células en la vena lateral o dorsal de la cola utilizando una jeringa de 1mL con aguja de calibre 2516.</li>
</ol>2. inmunización (Endo-libre OVA +/-adyuvante)
<ol><li>Colocar los ratones transplantados con OT-I (paso 1.7, figura 1) de las células en una cámara de anestesia y administrar isoflurano en oxígeno por una máquina de anestesia14.</li>
	<li>Cuando el ratón está totalmente dormido bajo anestesia, sacar de la cámara y afeitarse la piel del ratón en el área sobre los superficialis de los glúteos (zona lumbar lateral) mediante el uso de una máquina de corte de pelo eléctrico, con el fin de realizar una inyección limpia con una buena vista de la zona de aplicación.</li>
	<li>Inyectar 50 μl de la vacuna, s.c. en el área depilado con una aguja de calibre 25.</li>
</ol>3. aislamiento de linfocitos y de tinción para análisis de citometría de flujo
<ol><li>Aislamiento de linfocitos y esplenocitos<ol>
<li>Eutanasia a los ratones vacunados por CO2 inhalación seguida por dislocación cervical.</li>
		<li>Extraer los ganglios linfáticos de drenaje y distantes y bazo y colocar en cajas Petri separadas que contienen 100 μm poro malla tazas para lanzamiento de pulido y de la célula del tejido. Siga los pasos de 1.1.2 a 1.1.3.</li>
		<li>Decantar el sobrenadante después de centrifugación y vuelva a suspender los pellets de células en el mismo volumen de PBS del remanente (aproximadamente 100 μL).</li>
	</ol>
</li>
	<li>Tinción para análisis de citometría de flujo<ol>
<li>En un tubo de centrífuga de 15 mL, prepare una mezcla principal que contiene los anticuerpos tinción en las concentraciones que se muestra en la tabla 1, así que rinde 2 x concentrado mezcla de tinción. Prepare suficiente volumen de mezcla principal a 100 μl por muestra. Mezcle las células y el maestro mezcla 1:1 e incubar a 4° C por 30 minutos. Nota: El volumen final de la tinción por muestra debe 200 μl (100 μl de células pellet + 100 μl de la mezcla principal anticuerpo).</li>
		<li>Lavan las células dos veces por centrifugación como se describe en 1.1.3, agregar 10 mL de PBS, dos veces.</li>
		<li>Resuspenda las células en 0.5-1 mL de PBS para la adquisición y transferencia de la suspensión a los tubos de citómetro de flujo. Siempre mantenga las muestras en hielo y protegidos de la luz.</li>
	</ol>
</li>
</ol>4. citometría de flujo
<ol><li>Siempre prefiltro las muestras de células mediante filtros de 70-100 μm.</li>
	<li>Preparar solo tinción controles de compensación para los fluoróforos detallados en la tabla 1 (granos o células). Nota: Compensación debe ser realizada en citómetro o análisis software17,18,19 y aplicada a todas las muestras.</li>
	<li>Seguir la estrategia bloquea representada en la figura 2. Brevemente, poblaciones de la puerta en altura de dispersión hacia adelante frente a la zona de dispersión hacia adelante (figura 2A) y otra vez dispersión lateral amplia vs área de dispersión lateral (SSA, figura 2B) para excluir dobletes.</li>
	<li>Puerta de la población de figura 2B trazando BV 650 (auto-fluorescencia) vs FITC (CFSE) para discriminar la CFSE verdadero manchados células de células auto-fluorescente alta. Incluir granos o células teñidas con anticuerpos conjugados a 650 de BV en controles de compensación. Este diagrama (figura 2) de 650 BV vs FITC se utiliza para el OT de la puerta-me CFSE manchadas las células.</li>
	<li>Puerta de la población de figura 2 C Pe-Cy7 (Thy1.1 - CD90.1-) vs APC (CD8) para distinguir las células derivadas de donantes vs ratones receptores. Nota: Células derivadas de OT-I ratones donantes son Thy1.1+ (CD90.1), mientras que WT (C57BL/6, destinatarias) ratones las células derivadas son Thy1.2+ (CD90.2) (Figura 2D). Algunos laboratorios tienen OT-I ratones en un fondo de Thy1.2 y WT (C57BL/6) en un Thy1.1. En este caso tienes que usar un anticuerpo contra Thy1.2 para OT-célula de control.</li>
	<li>Volver a puerta CD8 de las células de población Thy1.1+ + trazando en una puerta que comprende 450 BV (CD4) y APC (CD8) (Figura 2E).</li>
	<li>Mostrar la población cerrada en Figura 2E mediante el uso de una pantalla de histograma de los CD8+ células mostrando CFSE (FITC, canal 530/15 - láser azul-), la puerta la población proliferada incluyendo intensidades de 102 hasta el nivel donde las poblaciones de control son (intensidades ~ 105, dependiendo de la eficacia de la tinción de CFSE y el ajuste de la tensión en el citómetro de flujo) (figura 2F).</li>
	<li>Hacer un puerta adicional (no se muestra en la figura 2) trazado marcador FITC vs L/D (450/20 canal, láser UV) con el fin de evaluar las células muertas en paralelo a la evaluación de la proliferación.</li>
	<li>Adquirir por lo menos 5000 eventos para los controles de compensación y 10,000 eventos (puerta E, figura 2) de las muestras en un citómetro de flujo. Ejecutar el citómetro de flujo de baja o media (no exceder las tasas de flujo de 20.000 eventos/s).</li>
</ol>

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Los autores no tienen nada que revelar.

Las vacunas modernas son idealmente composedof antígeno purificado y adyuvantes, con la posible adición de un sistema de entrega como liposomas, virus-como partículas, nanopartículas o vectores vivos. Un aspecto clave en el diseño de una vacuna es elegir el adyuvante adecuado según las necesidades clínicas. Parte del ámbito podría implicar favoreciendo una humoral y respuesta inmune celular (o ambos), la elección de un local frente a una respuesta inmune sistémica (o ambos) y el tipo de memoria que la vacuna d...

Los linfocitos T citotóxicos (CTL) que las vacunas son fundamentales intervenciones terapéuticas que se han desarrollado para luchar contra a ciertos tipos de cáncer1. También son importantes las vacunas profilácticas contra patógenos intracelulares2CTL. Por otra parte, CTL son uno de los pocos mecanismos de defensa inmunes funcionalmente activos en poblaciones de riesgo como neonatos3,4 quien también dependen de CTL para luchar contra la temprana vida infecciones5. En este sentido, las vacunas contra el Virus sincitial respiratorio (VSR) que fueron desarrolladas con un adyuvante que no provocan respuestas CTL (alumbre) resultaron en un fracaso de la vacuna lleva a serias complicaciones con infecciones en recién nacidos6. Estos efectos negativos de la vacunación pueden invertirse por una CD8+ de respuesta de la célula de T7. Previamente hemos demostrado que las citoquinas principales (tipo interferones de I) sacadas por algún estimulador de agonistas (aguijón) los genes de interferón son esenciales para las respuestas CTL generadas por estos adyuvantes8, en parte por la medición de la proliferación de OT-I T las células después de la vacunación y usar estos resultados como una medida de CTL inducen las capacidades observadas en extendieron vacunación horarios9. La medición de la proliferación de OT-I CD8+ T las células en un ratón receptor de tipo salvaje (WT) C57BL/6 por carboxyfluorescein succinimidyl tinte de éster (CFSE) dilución es una estimación robusta de la capacidad de los adyuvantes de una vacuna para generar reactividad cruzada de SIINFEKL, (el péptido inmuno-dominante de ovoalbúmina, óvulos). Variaciones de esta técnica están ampliamente utilizadas para la evaluación de la proliferación de OT-I CD8+ y OT II CD4+ T las células. Por ejemplo, se ha utilizado en la ausencia de las citoquinas (ratones KO) o para medir la eficacia de la vacuna después de memoria del antígeno en animales de peso. Ideamos un protocolo corto (4 días experimento) en el que después de la transferencia pasiva de OT CFSE-manchado-I CD8 células+ T, una vacunación subcutánea de (s.c.) que consiste en una dosis de 50 μg de huevos libres de endotoxinas complementado con adyuvantes de la prueba se administra (Figura 1). El seguimiento de los resultados 48 horas después de la vacunación proporciona una prueba confiable de la capacidad de los adyuvantes para generar respuestas CTL. Por esta estrategia, es posible evaluar la potencia de la respuesta inmune local en el nodo de linfa de drenaje después de la inmunización, así como la magnitud de la respuesta mediante la medición de la actividad CTL en el bazo (o los ganglios linfáticos distantes).

<table><tbody><tr><td>BD LSR Fortessa Cell Analyzer</td><td>BD</td><td>Special Order</td><td>Flow Cytometer</td></tr><tr><td>CFSE</td><td>Molecular Probes</td><td>C34554</td><td>Proliferation Dye</td></tr><tr><td>MojoSort Mouse CD8 T Cell Isolation Kit</td><td>Biolegend</td><td>480007</td><td>Magnetic Isolation Beads and antibodies for negative selection of untouched CD8 T cells.</td></tr><tr><td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation</td><td>Molecular Probes</td><td>L23105</td><td>Dead Cell Marker</td></tr><tr><td>CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), PE-Cyanine7</td><td>eBioscience</td><td>25-0900-82</td><td>antibody</td></tr><tr><td>APC anti-mouse CD8a Antibody</td><td>BioLegend</td><td>100712</td><td>antibody</td></tr><tr><td>BV421 Rat Anti-Mouse CD4</td><td>BD</td><td>740007</td><td>antibody</td></tr><tr><td>Z2 coulter Particle count and Size Analyzer</td><td>Beckman Coulter</td><td>9914591DA</td><td>Cell counter. Z2 Automated particle/cell counter</td></tr><tr><td>EndoGrade Ovalbumin (10 mg)</td><td>Hyglos(Germany)</td><td>321000</td><td>Ovalbumin endotoxin free tested.</td></tr><tr><td>Cell Strainer 100&amp;micro;m nylon</td><td>Corning</td><td>352360</td><td>Cell strainer (100 &amp;micro;m pore mesh cups).</td></tr><tr><td>Sample Vials</td><td>Beckman Coulter</td><td>899366014</td><td>Sample vials for Z2 automated counter</td></tr><tr><td>C57BL/6 mice (CD90.2)</td><td>Harlan (Rossdorf, Germany)</td><td></td><td>Company is now Envigo</td></tr><tr><td>OT-I (C57BL/6 background, CD90.1)</td><td>Harlan (Rossdorf, Germany)</td><td></td><td>Inbreed at our animal facility. Company from where adquired is now Envigo</td></tr><tr><td>FACS tubes</td><td>Fischer (Corning)</td><td>14-959-5</td><td>Corning Falcon Round-Bottom Polystyrene Tubes</td></tr><tr><td>Falcon 15 mL tubes</td><td>Fischer (Corning)</td><td>05-527-90</td><td>Falcon 15mL Conical Centrifuge Tubes</td></tr><tr><td>PBS (500 mL)</td><td>Fischer (Gibco)</td><td>20-012-027</td><td>Gibco PBS (Phosphate Buffered Saline), pH 7.2</td></tr><tr><td>Red lamp (heating lamp)</td><td>Dirk Rossmann GmbH (Germany)</td><td>405096</td><td>Heating infrred lamp (100 wats)</td></tr><tr><td>IsoFlo (Isoflurane)</td><td>Abbott Laboratories (USA)</td><td>5260.04-05.</td><td>Isoflurane anesthesic (250 mL flask).</td></tr><tr><td>&lt;strong&gt;Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber&lt;/strong&gt;</td><td>Parkland Scientific</td><td>V3000PK</td><td>Isoflurane anesthesia machine.</td></tr><tr><td>RPMI 1640 medium</td><td>Gibco (distributed by ThermoFischer)</td><td>11-875-093</td><td>Base medium with Glutamine (500 mL)</td></tr><tr><td>Pen-Strept antibiotic solution (Gibco)</td><td>Gibco (distributed by ThermoFischer)</td><td>15-140-148</td><td>Gibco Penicillin-Streptomycin (10,000 U/mL)</td></tr><tr><td>Fetal Bobine Serum (Gibco)</td><td>Gibco (distributed by ThermoFischer)</td><td>10082147</td><td>Fetal Bovine Serum, certified, heat inactivated, US origin</td></tr><tr><td>ACK Lysing Buffer (100 ml)</td><td>Gibco (distributed by ThermoFischer)</td><td>A1049201</td><td>Amonium Chloride Potasium (ACK) Whole Blood Lysis Buffer, suitable for erytrocyte lysis in spleen suspensions also</td></tr><tr><td>Plastic Petri Dishes</td><td>Nunc (distributed by ThermoFischer)</td><td>150340</td><td>60 x 15mm Plastic Petri Dish, Non-treated</td></tr><tr><td>Cell Clump Filter</td><td>CellTrics (Sysmex)</td><td>04-004-2317</td><td>CellTrics&amp;reg; 50 &amp;mu;m, sterile</td></tr></tbody></table>

rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development

La evaluación de las vacunas de subunidad moderna revela que la generación de anticuerpos neutralizantes es importante pero no suficiente para la selección del adyuvante. Por lo tanto, están urgente adyuvantes con capacidades inmuno-estimuladora humorales y celulares que son capaces de promover respuestas citotóxicas de los linfocitos (CTL) T. Así, el fiel seguimiento de candidatos de adyuvante que inducen reactividad cruzada y posteriormente aumentar la generación de CTL representa un paso crucial en el desarrollo de una vacuna. Aquí presentamos una aplicación de un método que utiliza SIINFEKL específicos (OT-I) las células T para controlar la presentación cruzada del modelo antígeno ovoalbúmina (OVA) en vivo en la presencia de candidatos de diferentes adyuvantes. Este método representa una prueba rápida para seleccionar adyuvantes con las mejores capacidades de reactividad cruzada. La proliferación de CD8+ T las células es la más valiosa indicación de reactividad cruzada y también es considerado como un correlato de presentación cruzada inducida por adyuvante. Esta característica puede ser evaluada en diversos órganos inmunes como los ganglios linfáticos y el bazo. También puede supervisar el grado de la generación de CTL, que dando ideas sobre la naturaleza de un local (nodo de linfa de drenaje principalmente) o una respuesta sistémica (distante de los ganglios linfáticos o bazo). Esta técnica además permite múltiples modificaciones para probar fármacos que pueden inhibir vías específicas de presentación cruzada y también ofrece la posibilidad de ser utilizado en diferentes cepas de ratones modificados genéticamente y convencionales. En Resumen, la aplicación que presentamos aquí será útil para los laboratorios de la vacuna en la industria o la academia que desarrollaran o modificar químicos adyuvantes de la vacuna investigación y desarrollo.

Para probar los tratamientos utilizando una combinación diferente de los adyuvantes (ADJ1 y ADJ2), hemos evaluado la capacidad de generación de CTL midiendo la proliferación de OT eventualmente transferido-I CD8+ T las células mediante citometría de flujo (figura 2). Para esto, hemos manchado previamente células aisladas de los ganglios linfáticos y bazo (tabla 1) drenaje. Mediante la medición de la proliferación de CD8<su...

Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Aquí presentamos una aplicación de una técnica inmunológica estándar (CFSE manchado OT-I proliferación) pretende monitorear rápidamente mediada por el adyuvante de linfocitos T citotóxicos (CTL) generación en vivo. Esta estimación rápida de la capacidad CTL es útil para el desarrollo de vacunas profilácticas contra patógenos intracelulares así como vacunas contra el cáncer terapéutico.

Rápida In Vivo evaluación de las capacidades de generación de linfocitos T citotóxicos de coadyuvante para el desarrollo de una vacuna

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

rapid-vivo-assessment-adjuvant-s-cytotoxic-t-lymphocytes-generation

Research

JoVE Journal

Medicine

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

8.6K vistas.  Helmholtz Centre for Infection Research. Aquí presentamos una aplicación de una técnica inmunológica estándar (CFSE manchado OT-I proliferación) pretende monitorear rápidamente mediada por el adyuvante de linfocitos T citotóxicos (CTL) generación en vivo. Esta estimación rápida de la capacidad CTL es útil para el desarrollo de vacunas profilácticas contra patógenos intracelulares así como vacunas contra el cáncer terapéutico.

Video: Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

Ensayo in vitro para evaluar el impacto de inmunoreguladoras Caminos de CD4 T función específica para el VIH efector celular

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Investigación

Inmunología e Infección

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

El uso de fluorescentes Arrays objetivo para la evaluación de las respuestas de células T<em&gt; En vivo</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Imaging Whole-animal y de citometría de flujo Las técnicas para T CD8 + respuestas celulares Análisis de antígeno-específicos después de nanopartículas Vacunación

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

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 ...

Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we investigated immunological effects of a novel lipid-DNA immunostimulant in nanoparticle form from different administration routes in the mouse: oral, intranasal, subcutaneous, footpad, intraperitoneal, and intravenous. These injections will directly influence the immune response, and harvesting lymphatic tissues and analysis of dendritic cell (DC) activation in the tissues are crucial parts of these evaluations. The extraction of mediastinal lymph nodes (mLNs) is important but quite complex because of the size and location of this organ. A stepwise procedure for harvesting the inguinal lymph node (iLN), mLN, and spleen and analyzing DC activation by flow cytometry is described.

Experimental procedures for the subsequent extraction of lymphatic tissues to test lymphoid dendritic cell activation are described after treatment of an immunostimulating nanomaterial.

Evaluación de inmunoestimulantes agente: Linfoide inyección y extracción de tejido dendrítico de la célula dependiente de la ruta de activación

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Confección de ensayos de citotoxicidad in vivo para estudiar inmunodominancia en respuestas de células T CD8+ específicas de tumores

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; in particular, some MHC I-restricted epitopes can induce effective cytotoxic T lymphocyte activity to clear tumor cells. Additionally, nasal administration is an effective and safe delivery technique for tumor vaccines due to its convenience and improved patient compliance. However, epitope peptides are unsuitable for nasal delivery because of their poor immunogenicity and lack of delivery efficiency. Nanoemulsions (NEs) are thermodynamically stable systems that can be loaded with antigens and delivered directly to the nasal mucosal surface. Ile-Lys-Val-Ala-Val (IKVAV) is the core pentapeptide of laminin, an integrin-binding peptide expressed by human respiratory epithelial cells. In this study, an intranasal self-assembled epitope peptide NE tumor vaccine containing the synthetic peptide IKVAV-OVA257-264 (I-OVA) was prepared by a low-energy emulsification method. The combination of IKVAV and OVA257-264 can enhance antigen uptake by nasal mucosal epithelial cells. Here, we establish a protocol to study the physicochemical characteristics by transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS); stability in the presence of mucin protein; toxicity by examining the cell viability of BEAS-2B cells and the nasal and lung tissues of C57BL/6 mice; cellular uptake by confocal laser scanning microscopy (CLSM); release profiles by imaging small animals in vivo; and the protective and therapeutic effect of the vaccine by using an E.G7 tumor-bearing model. We anticipate that the protocol will provide technical and theoretical clues for the future development of novel T cell epitope peptide mucosal vaccines.

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

Preparación, características, toxicidad y evaluación de la eficacia de la vacuna nasal autoensamblada para tumores de nanoemulsión in vitro e in vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; ...

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

In vitro Evaluación de la actividad celular del adyuvante de la vacuna de nanoemulsión Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

Determinación de la inmunogenicidad de la vacuna utilizando células dendríticas derivadas de monocitos bovinos

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...

An Immunological Technique to Monitor Adjuvant-Mediated Cytotoxic T Lymphocyte Generation

Source: Lirussi, D. et al., <a href="https://app.jove.com/v/57401">Rapid In Vivo&#160;Assessment of Adjuvant&#39;s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development.</a> J. Vis. Exp. (2018)
The video demonstrates an immunological method to monitor in vivo adjuvant-induced cytotoxic T lymphocyte or CTL production. A mouse is pre-injected with genetically engineered, proliferative dye-stained donor CD8+ T lymphocytes. Subcutaneous injection of ovalbumin with adjuvants generates cytotoxic CD8+ T lymphocytes that are identified post-harvesting through immunostaining and flow cytometry.

Una técnica inmunológica para monitorizar la generación de linfocitos T citotóxicos mediados por adyuvantes

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
All mice ...

Rápida In Vivo evaluación de las capacidades de generación de linfocitos T citotóxicos de coadyuvante para el desarrollo de una vacuna

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