This work was supported by funding received from the National Institutes of Health (1R01AI083269).

Los estudios murinos fueron aprobados por el Cuidado y Uso de Animales Comité Institucional (IACUC) de la Universidad de Carolina del Norte en Chapel Hill y todo cuidado de los animales estaba en conformidad con las directrices del IACUC. 1. Preparación del Recién Nacido y del adulto Thymi <ol><li> Preparar todos los reactivos y el equipo antes de la eutanasia de los ratones donantes. </li><li> Esterilizar los instrumentos quirúrgicos de esterilización en autoclave u otros métodos apropiados. Todos los procedimientos quirúrgicos deben realizarse bajo una campana de flujo laminar para mantener las condiciones estériles y evitar la contaminación. Montar las herramientas necesarias para la extracción de timos de ratones donantes. </li><li> Llene un plato de 60 mm con estéril 1x PBS (pH 7,4) y colocar en hielo dentro de la campana. Esto se utilizará para almacenar brevemente timos extirpados de ratones donantes antes del trasplante. </li><li> De acuerdo con las normas éticas, la eutanasia a los ratones donantes recién nacidos por decapitación y los ratones donantes adultos por asfixia de CO 2 seguido por dislocación cervical. </li><li> Para removal del timo: Coloque el ratón en una posición reclinada dorsal sobre una toalla de papel absorbente estéril y rociar con etanol al 70% antes de hacer una incisión. </li><li> Exponer la cavidad abdominal y torácica haciendo una incisión en la línea media a través de la piel. Doble la piel sobre el pecho y extremidades anteriores para revelar la cavidad torácica. </li><li> Haga dos incisiones laterales a través del diafragma y la caja torácica para exponer el mediastino superior y anterior de la cavidad torácica. El timo debe ser visible como dos lóbulos blancos inmediatamente por encima y adyacente al corazón. </li><li> Desmenuzar el tejido conectivo que rodea el timo con unas pinzas finas, asegurándose de no perturbar la cápsula. Mientras mantiene de nuevo la caja torácica con fórceps, utilizar un segundo par de pinzas para extraer los dos lóbulos del timo mediante la colocación de pinzas curvas debajo del órgano y tirando verticalmente. Esto se puede hacer usando un microscopio de disección para extraer timos de ratones recién nacidos. </li><li> Coloque el timo en el 60 mm dish que contiene 1x PBS estéril (pH 7,4) en hielo y los lóbulos tímicos separados por el corte a través del istmo conectivo. Retire cualquier residuo de los lóbulos tímicos asegurándose de no dañar la cápsula, y cortar el timo en el número apropiado de secciones para el trasplante. </li><li> No manipular el timos obtenido a partir de ratones recién nacidos. NOTA: Se utiliza para un máximo de dos ratones receptores (1 lóbulo por destinatario). </li><li> Transplante las timos obtenido a partir de ratones adultos en un máximo de 4-6 ratones receptores. El uso de un par de pinzas, sujete cuidadosamente lóbulo tímico un adulto, como se muestra en la Figura 1, cortar el timo en tres secciones iguales utilizando tijeras quirúrgicas. </li><li> Repita los pasos 1.3 a 1.4 para cada ratón donante. Con el fin de limitar el tiempo timos están expuestos, preparar timo donante 1 a la vez. </li></ol> 2. Implantación timo bajo la cápsula renal <ol><li> Montar el equipo de pre-esterilizados listado en la Tabla 1. Aséptica adecuadotécnica debe ser utilizada durante el curso del procedimiento para prevenir la exposición de trasplante-receptores a las herramientas o reactivos contaminados. </li><li> Antes del trasplante, pesar y etiquetar cada ratón receptor. </li><li> Configure el microscopio de disección y el circuito de la anestesia en la campana de flujo laminar. </li><li> El uso de una máquina de afeitar eléctrica, afeitarse el lado izquierdo de la ratón receptor y asegurar sin pelo permanece alrededor de la zona utilizada para hacer la incisión. </li><li> Encienda vaporizador isofluorano y anestesiar el ratón utilizando una dosis de entre 1-2%. Determinar anestesia adecuada antes de comenzar el procedimiento quirúrgico mediante la comprobación de una ausencia de reflejo siguiente pizca dedo del pie. </li><li> Después de que el ratón está anestesiado adecuadamente, aplicar el ungüento veterinaria a los ojos del ratón para evitar la sequedad y preparar el ratón para el trasplante como sigue: </li><li> Coloque el ratón bajo el microscopio de disección en posición decúbito lateral derecho para que el lado afeitado quede hacia arriba. Starting en el centro de la zona quirúrgica, dispensar en un movimiento circular usando una pipeta de transferencia desechable 70% de etanol, seguido de povidona-yodo (betadine). Repetir el tratamiento con etanol / betadine 3 veces antes de hacer una incisión. </li><li> Usando tijeras de disección hacen una incisión en el flanco 1-2 cm directamente sobre el riñón. </li><li> El uso de pinzas medianas agarrar el tejido conectivo adyacente al riñón y levantar suavemente el riñón de modo que se encuentra encima de la musculatura. Para mantener el riñón expuesto y en su lugar sobre la musculatura, inserte un brazo de fórceps medio por debajo del riñón, prestando especial atención a no perturbar el hueso ilíaco renal. </li><li> Con el fin de evitar la desecación del tejido, irrigar el riñón con 1x PBS estéril (pH 7,4) durante cada paso hasta que la manipulación de la cápsula renal y el riñón es completa. </li><li> Usando unas pinzas finas pellizcan y levantar la cápsula cerca del borde del riñón más distal a las glándulas suprarrenales para separarlo del riñón. </li><li> Utilice un calibre 18aguja para hacer una incisión lo suficientemente grande como para insertar la pieza preparada de timo donante (Figura 2A). Asegúrese de mantener la incisión capsular tan pequeño como sea posible para evitar la dislocación del injerto con el tiempo. </li><li> Mientras se mantiene la cápsula se apartó desde el riñón, utilizar un segundo par de pinzas finas para insertar el injerto debajo de la cápsula y empujar el timo lo más adelante de la incisión capsular como sea posible (Figura 2B). </li><li> Retire las pinzas de debajo de los riñones, y volver suavemente el riñón nuevo en su lugar a través de la incisión. </li><li> Cierre la musculatura suturando la pared peritoneal y aplicar betadine una vez cerrada. Después de sutura es completa, aplicar clips de la herida para cerrar la dermis y aplicar betadine a la zona circundante. </li><li> Devuelva el ratón a una jaula que se ha calentado utilizando una lámpara de calor. </li><li> Monitorear cada ratón postoperatorio hasta que recupera la conciencia plena y la movilidad y no coloque tha ratonest no se han recuperado después de la anestesia en una jaula con otros animales. </li><li> Tratar ratones con fármacos de control del dolor post-operatorio, según sea necesario. Proporcionar ratones con analgésicos post-operatorias tales como el acetaminofeno en el agua de bebida a una concentración de 1,6 mg / ml. </li><li> Repita los pasos 2.3 a 2.9 por cada ratón receptor. </li><li> Asegúrese de que los ratones retorno a la actividad normal dentro de 1 hora después de la cirugía. Monitor de peso post-operatorio, habilidades motoras, así como la actividad de beber y la alimentación de cada ratón para determinar las complicaciones que surgen como resultado del procedimiento de trasplante. NOTA: la pérdida de peso post-operatorio, en comparación con el peso preoperatoria, así como la movilidad alterada o el fracaso para comer o beber puede indicar complicaciones. </li><li> Monitor de reconstitución de células T en la sangre periférica por sangrado ratones a través de la cola nick seguido de separación de linfocitos usando Lympholyte medios de separación de células de acuerdo con las especificaciones del fabricante. Lograr characteriza células Tción por tinción linfocitos con anticuerpos conjugados con fluorescencia específicos para marcadores de células T CD3, CD4, y CD8, así como un discriminador de muertos en vivo, tales como un éster de succinimidilo activado por fluorescencia 8. Análisis como se muestra en la Figura 3 se puede lograr a través de gating en interiores, de células vivas, y las células CD3 positivas. </li></ol>

<ol>
	<li>Kappler, J. W., Roehm, N., Marrack, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+tolerance+by+clonal+elimination+in+the+thymus.">T cell tolerance by clonal elimination in the thymus.</a> Cell. 49 (2), 273-280 (1987).</li><li>Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Journey+through+the+thymus:+stromal+guides+for+T-cell+development+and+selection.">Journey through the thymus: stromal guides for T-cell development and selection.</a> Nat. Rev. Immunol. 6, 127-135 (2006).</li><li>Klein, L., Kyewski, B., Allen, P. M., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+and+negative+selection+of+the+T+cell+repertoire:+what+thymocytes+see+(and+don&#8217;t+see).">Positive and negative selection of the T cell repertoire: what thymocytes see (and don&#8217;t see).</a> Nat. Rev. Immunol. 14 (6), 377-391 (2014).</li><li>Schwartz, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+immunologic+self-tolerance.">Acquisition of immunologic self-tolerance.</a> Cell. 57 (7), 1073-1081 (1989).</li><li>Kishimoto, H., Sprent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+defect+in+central+tolerance+in+NOD+mice.">A defect in central tolerance in NOD mice.</a> Nat. Immunol. 2 (11), 1025-1031 (2001).</li><li>Anderson, G., Jenkinson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+central+tolerance+with+reaggregate+thymus+organ+cultures.">Investigating central tolerance with reaggregate thymus organ cultures.</a> Methods. Mol. Biol. 380, 185-196 (2007).</li><li>He, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymic+development+of+autoreactive+T+cells+in+NOD+mice+is+regulated+in+an+age-dependent+manner.">Thymic development of autoreactive T cells in NOD mice is regulated in an age-dependent manner.</a> J. Immunol. 191 (12), 5858-5866 (2013).</li><li>Stewart, C. C., Stewart, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+6.+Immunophenotyping.">Chapter 6. Immunophenotyping.</a> Current Protocols in Cytometry. , (2001).</li><li>Rice, H. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymic+transplantation+for+complete+DiGeorge+syndrome:+medical+and+surgical+considerations.">Thymic transplantation for complete DiGeorge syndrome: medical and surgical considerations.</a> J. Pediatr. Surg. 39 (11), 1607-1615 (2004).</li><li>Markert, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+thymus+tissue+in+complete+DiGeorge+Syndrome.">Transplantation of thymus tissue in complete DiGeorge Syndrome.</a> N. Engl. J. Med. 341, 1180-1189 (1999).</li><li>Market, M. L., Devlin, B. H., McCarthy, 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=Thymus+transplantation.">Thymus transplantation.</a> Clin. Immunol. 135 (2), 236-246 (2010).</li><li>Wells, A. D., Li, X., Strom, T. B., Turka, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+peripheral+T-cell+depletion+in+transplantation+tolerance.">The role of peripheral T-cell depletion in transplantation tolerance.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 356 (1409), 617-623 (2001).</li><li>Albuquerque, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+FOXN1-Deficiency+is+associated+with+ab+double-negative+and+FoxP3++T-cell+expansions+that+are+distinctly+modulated+upon+thymic+transplantation.">Human FOXN1-Deficiency is associated with ab double-negative and FoxP3+ T-cell expansions that are distinctly modulated upon thymic transplantation.</a> PLoS One. 7 (5), e37042 (2012).</li><li>Chinn, I. K., Milner, J. D., Scheinberg, P., Douek, D. C., Markert, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymus+transplantation+restores+the+repertoires+of+forkhead+box+protein+3+(FoxP3)++and+FoxP3-+T+cells+in+complete+DiGeorge+anomaly.">Thymus transplantation restores the repertoires of forkhead box protein 3 (FoxP3)+ and FoxP3- T cells in complete DiGeorge anomaly.</a> Clin Exp Immunol. 173 (1), 140-149 (2013).</li><li>Savino, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thymus+is+a+common+target+organ+in+infectious+diseases.">The thymus is a common target organ in infectious diseases.</a> PLoS Pathog. 2 (6), e62 (2006).</li></ol>

The authors do not have any competing financial interests to disclose.

Events regulating thymic selection are poorly understood. Recent studies have demonstrated ontogenic changes in the efficiency of selection. Coupling this approach with different transgenic thymus donor and recipient mice will further facilitate studies to identify the events and parameters that regulate thymic selection. Notably, thymic transplantation is used for the treatment and management of T cell and thymic disorders seen for instance in athymic infants lacking functional T cells and in patients with DiGeorge synd...

El timo es un órgano en el que los eventos críticos en el desarrollo de células T producen 1. Timocitos Residentes, sobre reordenación del receptor de células T (TCR) α y β genes, se someten a una serie de interacciones con lymphostromal y células presentadoras de antígeno (APC) en las regiones corticales y medulares del timo 2. Selección positiva tímico está mediada por células epiteliales corticales del timo (TEC) para producir los timocitos que reconocen péptidos antigénicos en el contexto de anfitrión complejo mayor de histocompatibilidad (MHC) de clase I y II moléculas 2-3. La posterior selección negativa tímica implica la purga de los timocitos autorreactivos, impulsado por una interacción con TEC medular o células dendríticas (DC) que presentan péptidos derivados de auto-proteínas unidas por MHC de clase I y II moléculas 3. El resultado final de estos procesos es el establecimiento de un grupo de células maduras T CD4 + y CD8 + capaces de responder a un amplio espectrode antígenos extraños mientras que exhiben una reactividad mínima a proteínas propias 4. La eficiencia de eventos de selección tímica está influenciada por una serie de factores, incluyendo la maduración tímica, la frecuencia de TEC medular y cortical, composición subconjunto de tímico DC, y la fuente de los precursores tímicos 3. En particular, la selección tímica aberrante puede resultar en autoinmunes 5 o inmunodeficientes patologías, que surgen de deterioro de la selección negativa o positiva, respectivamente. Los eventos moleculares que regulan la selección tímica, sin embargo, son poco conocidos. In vitro enfoques tales como cultivos de órganos timo reaggregate (RTOC) 6, han demostrado ser útiles para el análisis de los acontecimientos básicos asociados con la selección del timo, pero no para recapitular completamente la dinámica del curso en los eventos in vivo. Como resultado, este enfoque basado en el trasplante de timo se estableció a mejores eventos de selección de timocitos estudio in vivo 7 </sarriba&gt;. Este protocolo describe el trasplante de timos de ratones donantes recién nacidos y adultos en ratones receptores SCID inmunodeficientes. Esta técnica permite el estudio de los mecanismos que regulan la selección tímica positivo y negativo, así como la producción tímica de diversos subconjuntos de células T durante la ontogenia. Más recientemente, este enfoque ha sido utilizado para demostrar que la eficiencia de la selección tímica se limita temprano después del nacimiento en ratones conduce a un mayor desarrollo de las células T autorreactivas, y un reducido repertorio de células T específicas para antígenos extraños 7.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Decon Laboratories</td>
			<td>2705HC</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>GIBCO</td>
			<td>14190-144</td>
			<td>pH 7.2</td>
		</tr>
		<tr>
			<td>Betadine Solution</td>
			<td>Purdue Products</td>
			<td>67618-150-17</td>
			<td></td>
		</tr>
		<tr>
			<td>18 gauge needle</td>
			<td>BD</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures (1.0 metric)</td>
			<td>Ethicon</td>
			<td>J493G</td>
			<td>18&#34; (45cm)</td>
		</tr>
		<tr>
			<td>AUTOCLIP Wound Clips (9mm)</td>
			<td>Clay Adams&#174; Brand</td>
			<td>427631</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Fisher Scientific</td>
			<td>13-711-20</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr>
			<td>Mouse anti-CD3 Ab</td>
			<td>eBioscience</td>
			<td>11-0031-85</td>
			<td>Clone: 1452C11</td>
		</tr>
		<tr>
			<td>Mouse anti-CD4 Ab</td>
			<td>eBioscience</td>
			<td>48-0042-82</td>
			<td>Clone: RM4-5</td>
		</tr>
		<tr>
			<td>Mouse anti-CD8 Ab</td>
			<td>eBioscience</td>
			<td>25-0081-82</td>
			<td>Clone: 53-6.7</td>
		</tr>
		<tr>
			<td>Lancet</td>
			<td>Medipoint</td>
			<td>Goldenrod 4mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Orange Succinimidyl Ester, Triethylammonium Salt</td>
			<td>Invitrogen</td>
			<td>P30253</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well round botton polypropylene plates</td>
			<td>Corning</td>
			<td>3365</td>
			<td></td>
		</tr>
		<tr>
			<td>1.2mL polypropylene cluster tubes</td>
			<td>Corning</td>
			<td>4401</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL polypropylene round-bottom tubes</td>
			<td>BD</td>
			<td>352002</td>
			<td></td>
		</tr>
		<tr>
			<td>40uM Nylon Cell Strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Solution, EM Grade</td>
			<td>Electron Microscopy Services</td>
			<td>15710</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Puralube Optical Ointment</td>
			<td>Fisher Scientific</td>
			<td>NC0138063</td>
			<td></td>
		</tr>
		<tr>
			<td>Lympholyte</td>
			<td>Cedarlane</td>
			<td>CL5030</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture dish</td>
			<td>Corning</td>
			<td>430196</td>
			<td>60mm x 15mm, Sterile</td>
		</tr>
	</tbody>
</table>

isolation and transplantation of different aged murine thymic grafts.

Los mecanismos que regulan la eficacia de la selección tímica permanecen mal definidos. El método presentado aquí permite en los análisis in vivo de el desarrollo y selección de las células T específicas para antígenos propios y extraños. El enfoque implica la implantación de injertos tímicos derivados de diversos ratones de edad en receptores SCID inmunodeficientes. Durante un período relativamente corto de tiempo los destinatarios están completamente reconstituidos con células T derivadas de injerto de timo implantado. Sólo timocitos siembra el timo en el momento de aislamiento se someten a selección y se desarrollan en células T maduras. Como tal, los cambios en la naturaleza y la especificidad de las células T injertadas como una función de los acontecimientos del timo dependientes de la edad pueden ser evaluados. Aunque se requiere experiencia técnica para el trasplante de timo éxito, este método proporciona una estrategia única para estudiar in vivo una amplia gama de patologías que se deben a o un resultado de la función y / o homeostas tímico aberrantees.

El éxito de este procedimiento es dependiente de mínimo trauma quirúrgico, así como el posicionamiento preciso del injerto por debajo de la cápsula renal. El injerto tímico se debe cortar para asegurar apropiadamente dimensionado secciones tímicos para posterior trasplante como se muestra en la Figura 1. Siguiendo el esquema de la Figura 1, los timos de ratones recién nacidos o adulto se puede utilizar para el trasplante subcapsular éxito con resultados injerto de células T co...

Watch this Scientific Journal Video about Isolation and Transplantation of Different Aged Murine Thymic Grafts. at JoVE.com

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

Aislamiento y Trasplante de diferentes Aged murinos tímicos injertos.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

isolation-and-transplantation-of-different-aged-murine-thymic-grafts

Research

JoVE Journal

Immunology and Infection

13.7K vistas.  University of North Carolina at Chapel Hill. This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo. 

Video: Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Modelo murino de CD40-activación de las células B

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Investigación

Inmunología e Infección

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

El examen de selección tímica positivo y negativo por citometría de flujo

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

El aislamiento, tratamiento y análisis de células gingivales murinos

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

El aislamiento, la identificación y purificación de células epiteliales del timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Aislamiento de células estromales murinas en nódulos linfáticos

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Aislamiento y la inyección intravenosa de murino médula ósea derivados monocitos

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

El aislamiento y la citometría de flujo Caracterización de murino Small intestinal linfocitos

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

Preparación y aplicaciones de Organotípicos tímico Slice culturas

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Aislamiento y activación de los linfocitos murinos

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Murino de grosor completo Trasplante de Piel

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...