This work was supported by the Interdisciplinary Center for Clinical Research (IZKF) M&#252;nster (SEED 03/12, SB), Deutsche Forschungsgemeinschaft (SFB TR128, TP B6 to S.G.M. ME3283/2-1 to S.G.M.) and by Innovative Medizinische Forschung, M&#252;nster (I-BI111316, SB and SGM).

Todos los experimentos con ratones se deben realizar de acuerdo con las directrices del respectivo comité de cuidado de los animales y el uso institucional. 1. Comentarios generales para experimentos con ratones <ol><li> Mantenga los ratones en condiciones libres de patógenos y les permitirá el acceso a alimentos y agua ad libitum. NOTA: Es importante la utilización de ratones por edad y sexo en los grupos experimentales porque los patrones inmunológicos pueden variar con la edad y el género. </li></ol> 2. Las células T específicas de OVA Preparación y activación de CD8 + (OT-I) <ol><li> Realice la estimulación de las células T OT-I como se describe a continuación 5 días antes de la preparación de cortes de cerebro. NOTA: 5 x 10 5 efectoras CD8 + activadas OT-I células T (células T CD8 + de ratones transgénicos que reconocen sólo el OVA 257-264 péptido en el contexto de moléculas MHC-I) se necesitan por rebanada La concentración 5 x 10 5 fue elegido experimentoaliado como uno óptima para favorecer una buena interacción célula-célula una vez que las células comienzan a migrar a la rebanada y, al mismo tiempo, la cantidad de células no es demasiado alta para inducir una reacción exagerada de una sola célula lo que permite contar 8,18. </li><li> Preparar el medio para el cultivo de células T OT-I. Suplemento 500 ml de DMEM con 5% de suero de ternera fetal (FCS), 10 mM HEPES, 2 mM L-glutamina, 50 micras 2-mercaptoetanol, 1% de aminoácidos no esenciales y 25 mg / ml de gentamicina. Almacenar el medio a 4 ° C hasta su uso. </li><li> Retirar el bazo de OT-I ratones transgénicos como por referencia 16. Transferirlos al medio preparado. </li><li> Generar suspensión de células individuales por maceración bazos a través de un filtro de 70 micras y se centrifuga la suspensión a 300 xg durante 5 min, 4 ° C. </li><li> Incubar suspensión de células con 5 ml de amonio-cloruro de potasio (ACK) tampón (150 mM NH 4 Cl, 10 mM KHCO3, EDTA 0,1 mM, pH 7,3) durante 5 min para lisar las células rojas de la sangre. </li><li> Deje de incubatien 10 ml con medio y se centrifuga de nuevo como se describió anteriormente. </li><li> Diluir la suspensión celular en medio y calcular números. Células de la placa a una densidad de 3 x 10 7 células / pocillo en una placa de 12 pocillos y se les prime por incubación con OVA 257-264 (SIINFEKL; 1 nM) y la interleucina 2 (IL-2) (500 UI / ml) durante 5 días. </li><li> Después de 4 días, añadir IL-2 a una concentración de 500 UI / mL de nuevo. </li><li> Después de la estimulación, purificar OT-I células T de suspensión celular mediante el uso de un ratón basado selección negativa kit de aislamiento de células T CD8 +, siguiendo las instrucciones del fabricante. La purificación se basa en el agotamiento de todas las células no-CD8 + mediante el uso de un cóctel de anticuerpos contra CD4, CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, MHC Clase II, Ter-119, y TCRγ / δ que luego son desechados por el uso de columnas magnéticas para la elución de las células T CD8 + purificados. NOTA: Los pasos críticos en este procedire se indican por los fabricantes: es importante que la reacción implica sólo células individuales debido a la presencia de grupos podría bloquear la columna durante la etapa de elución; contando de la celda antes de añadir el cóctel Byotin es importante para el grado de pureza de la muestra y el procedimiento se debe realizar en hielo para minimizar los enlaces de anticuerpos no específicos. </li></ol> 3. Preparación de rodajas de cerebro agudo y co-cultivo con células T OT-I <ol><li> Antes de la preparación de cerebral aguda rebana 19, preparar la solución salina placedine fisiológica enfriada con hielo usando sacarosa 200 mM, tubos de 20 mm, 2,5 mM de KCl, 1,25 mM de dextrosa NaH 2 PO 4, 10 mM MgSO 4, 0.5 CaCl 2 y 10 mM y el líquido cefalorraquídeo artificial (ACSF) utilizando NaCl 125 mM, 2,5 KCl, 1,25 NaH 2 PO 4, 24 mM NaHCO 3, 2 mM MgSO 4, 2 mM CaCl 2, 10 mM de dextrosa. Ajuste el pH de cada solución a 7,35 por using NaOH. Antes de ajustar el pH de ACSF, solución debe burbujeó con una mezcla de 95% O 2 y 5% de CO 2. </li><li> Pre-enfriar las soluciones. </li><li> Anestesie 8 - 10 semanas de edad ratones (por ejemplo, ratones C57BL / 6 como control o ratones transgénicos ODC-OVA 17 con la inhalación usando 5,0% de isoflurano, 5,0% de halotano o inyectar 100 mg / kg de ketamina y 10 mg / kg de peso corporal a través de xilazina ip Espere para la anestesia y utilizar una pizca dedo del pie para evaluar el nivel de anestesia y decapitar a la vez. La eutanasia a los ratones como por el cuidado de animales institucional y utilizar criterios del comité para la eutanasia. </li><li> Ponga el ratón en posición ventral. Desinfectar y cortar el cuero cabelludo sagital. Abra el hueso craneal sagital, retire el cerebro rápidamente con la ayuda de una cuchara y fijarlo en el plato de un vibratome utilizando pegamento. </li><li> Llene la placa con la solución salina preparada placedine enfriado con hielo fisiológica. </li><li> Cortar 300 micras cortes coronales con la vibratome. NOTA: Este procedimiento se conocen para producir muestras de tejido intactas viables adecuados para estudios celulares funcionales dentro de un intervalo de tiempo de al menos 8 hr 19-21. En efecto, después de este período de tiempo, ya partir después de las 6 horas, la preparación muestra una menor calidad, es decir, cambios en las propiedades básicas y funcionales como la acción potencial de generación y propagación. </li><li> Después de seccionar, transferir una rebanada de inmediato en cada pocillo de una placa de 12 pocillos lleno de ACSF. </li><li> Añadir con cuidado 5 x 10 5 células OT-I T por rebanada. </li><li> Incubar las rebanadas para un máximo de 8 horas en una incubadora (37 ° C, 5% de CO 2). </li><li> Después del período de incubación, las rebanadas de cosecha e integrarlos mediante OCT compuesto tejido-tek y congelarlos en nitrógeno líquido. Almacenar a -20 ° C para otros estudios histológicos a por ejemplo, evaluar la estructura neuronal (por ejemplo, usando anti-MAPII o anticuerpos anti-sinaptofisina) o apoptosis (por ejemplo, utilizando anti caspasa-3-anticuerpos y anti-NeuN contracuerpos) de acuerdo con procedimientos estándar. </li></ol>

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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=A+pathogenic+role+for+myelin-specific+CD8(+)+T+cells+in+a+model+for+multiple+sclerosis.">A pathogenic role for myelin-specific CD8(+) T cells in a model for multiple sclerosis.</a> J Exp Med. 194 (5), 669-676 (2001).</li><li>McPherson, S. W., Heuss, N. D., Roehrich, H., Gregerson, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bystander+killing+of+neurons+by+cytotoxic+T+cells+specific+for+a+glial+antigen.">Bystander killing of neurons by cytotoxic T cells specific for a glial antigen.</a> Glia. 53 (5), 457-466 (2006).</li><li>Hogquist, K. 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A., Konnerth, A., Sakmann, B., Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thin+slice+preparation+for+patch+clamp+recordings+from+neurones+of+the+mammalian+central+nervous+system.">A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system.</a> Pflugers Arch. 414 (5), 600-612 (1989).</li><li>Meuth, S. 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The authors declare that they have no competing financial interests.

Diferentes modelos animales se han descrito en las últimas décadas para abordar las características patológicas de trastornos desmielinizantes inflamatorias como la esclerosis múltiple. En vivo modelos de ratón y de rata son ampliamente utilizados para imitar las características fisiopatológicas de la enfermedad, es decir, el análisis de las consecuencias de los procesos de desmielinización y remielinización y de episodios entremezcladas de inflamación y neurodegeneración. Sin embargo, sólo un enf...

Death of oligodendrocytes and destruction of the myelin sheath accompanied by loss of neurons and axons are typical pathological findings in grey matter lesions in individuals suffering from multiple sclerosis (MS)1,2. Cortical lesions can be divided so far in three different subtypes2: subpial, intracortical and leukocortical lesions. In comparison to white matter plaques, infiltrates are characterized by a predominance of CD8+ T-cells, suggesting their possible decisive role in grey matter inflammation3. Furthermore, oligoclonal expansions in blood, cerebrospinal fluid (CSF) and within inflammatory lesions can be found for CD8+ T-cells themselves4-6. 
In line with this, it is assumed that CD8+ T-cells may be specific for different myelin proteins7,8. Indeed, CD8+ T-cells are found near oligodendrocytes and myelin sheaths9,10 that show MHC I expression11 and might therefore be responsible for the loss of the myelin sheath. This process is often seen together with extensive &#8216;&#8216;collateral&#8217;&#8217; neuronal and axonal damage within the central nervous system (CNS) grey matter1,2. In fact, direct and indirect death of oligodendrocytes and neurons is induced by CD8+ T-cells via two different mechanism: (i) cell membrane swelling and rupture due to the formation of cytotoxic granules following the release of perforins and granzymes and (ii) ligation to the Fas receptors or exposition of FasL on their surface8,12,13.
Different types of animal models are established to study the underlying mechanism of the mentioned processes. In this respect, primed CD8+ T-cells specific for autoantigens with induced expression in CNS glial cells, like oligodendrocytes or astrocytes, can be adoptively transferred to analyse &#8216;&#8216;collateral&#8217;&#8217; neuronal and axonal death in grey matter subsequently14,15. To perform such in vivo experiments is a big help to mimic some pathophysiological aspects of MS, however, this approach is not suited to resolve the underlying mechanism and kinetics of axonal damage and neuronal apoptosis.
To overcome these restrictions, an ex vivo approach was established to study the mechanisms and time course of neuronal cell death following a oligondendrocytes-directed CD8+ T-cell attack. Since only oligodendrocytes and therefore myelin sheath production should be targeted by immune cells, MHC class-I-restricted, ovalbumin (OVA)-reactive OT-I Tcells are used16. These cells are subsequently transferred into brain slices obtained from mice selectively expressing OVA in oligodendrocytes (ODC-OVA mice)17.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:187px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:264px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">12-Well-plate</td>
			<td style="width:187px;">Corning</td>
			<td style="width:119px;">3513</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">2-Mercaptoethanol</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">31350-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">2-Methylbutan</td>
			<td style="width:187px;">Roth</td>
			<td style="width:119px;">3927.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">70 &#181;m strainer</td>
			<td style="width:187px;">Falcon</td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">CaCl2</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.02382.0500</td>
			<td>calcium chloride</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">CD8+-isolation kit</td>
			<td style="width:187px;">Miltenyi Biotech</td>
			<td style="width:119px;">130-090-859</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">D(+)-glukose</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.08337.1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">DMEM</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">31966-021</td>
			<td>warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">EDTA</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:119px;">E5134</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">FCS</td>
			<td style="width:187px;">PAA Laboratories</td>
			<td style="width:119px;">A15-151</td>
			<td>fetal calve serum</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">gentamicin</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">HEPES 1M</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">15630-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">IL-2</td>
			<td style="width:187px;">Peprotech</td>
			<td style="width:119px;">212-12</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Isofluran</td>
			<td style="width:187px;">Abbott</td>
			<td style="width:119px;">05260-05</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">KCl</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.04933.0500</td>
			<td>potassium chloride</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">KHCO3</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:119px;">P9144</td>
			<td>potassium hydrogen carbonate</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">L-Glutamine</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">35050-038</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">MgSO4</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.05886.0500</td>
			<td>magnesium sulfate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">NaCl</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:119px;">31434</td>
			<td>sodium chloride</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">NaH2PO4 * H2O</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.06346.0500</td>
			<td>sodium hydrogen phosphate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">NaHCO3</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.06329.0500</td>
			<td>sodium hydrogen carbonate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">NaOH</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.09137.1000</td>
			<td>sodium hydroxide</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">NH4Cl</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:119px;">213330</td>
			<td>ammonium chloride</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Non Essential Amino Acid</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">OVA (257-264)</td>
			<td style="width:187px;">Genscript</td>
			<td style="width:119px;">RP10611</td>
			<td>ovalbumin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">PIPES</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:119px;">P6757</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Sukrose</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.07687.1000</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Tissue-Tek OCT</td>
			<td style="width:187px;">Sakura</td>
			<td style="width:119px;">4583</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo model of an oligodendrocyte-directed t-cell attack in acute brain slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

Después de la incubación de secciones de cerebro con células T de oligodendrocitos dirigida CD8 +, oligodendrocitos, así como las neuronas sufren apoptosis (Figuras 2A y 1C, respectivamente). Signos histológicos de la apoptosis (por ejemplo, la caspasa-3, Tunel) pueden ser detectados más temprano después de 3 horas de incubación. El período de incubación no debe ser de más de 8 horas con el fin de garantizar una buena calidad de la preparación y resultados reproducibles. ...

Watch this Scientific Journal Video about An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices at JoVE.com

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Una<em&gt; Ex vivo</em&gt; Modelo de un ataque de células T Oligodendrocyte dirigida en Rebanadas cerebral agudo

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

an-ex-vivo-model-an-oligodendrocyte-directed-t-cell-attack-acute

Research

JoVE Journal

Immunology and Infection

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

6.8K vistas.  University of Münster. To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Video: An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Ex vivo de imágenes de células T en los ganglios linfáticos murino Rebanadas con campo amplio y microscopios confocal

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Investigación

Inmunología e Infección

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

La inducción de la enfermedad de injerto contra huésped y<em&gt; En Vivo</em&gt; T Monitoreo Celular Utilizando un modelo murino MHC de concordancia

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

Aislamiento y<em&gt; Ex Vivo</em&gt; Cultura de Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; Las células γδ T, una célula αβT Progenitor Extrathymic

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Establecimiento de un programa de instalación de alto rendimiento para la detección de pequeñas moléculas que modulan la c-di-GMP Señalización en el<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Un<em&gt; In Vitro</em&gt; Modelo de la barrera sangre-cerebro mediante Espectroscopia de Impedancia: Un enfoque en la interacción de células T de la célula endotelial

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

Aislamiento de la población lateral en la leucemia linfoblástica aguda inducida por linfocitos T de Myc en peces cebra

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

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 ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Un modelo de ratón In Vivo a medida ingenuo CD4 células T activación, proliferación y diferenciación de Th1 inducida por las células dendríticas derivadas de la médula ósea

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Un modelo de cultivo primario de células Bursal de pollo Ex Vivo para estudiar la patogenesia de la enfermedad Bursal infecciosa Virus

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

Cuantificación de basados en citometría de flujo multicolor de las mitocondrias y los lisosomas en las células T

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...