We thank Dr. Alexander Flugel (Martinsried, Germany) for providing us with the GFP-labeled, ovalbumin-specific T cell line used in this protocol.

1. Stimulation of the ovalbumin-specific T lymphocytes
Resting ovalbumin-specific T cells are stimulated with ovalbumin in the presence of irradiated Lewis rat thymocytes (30 Gy) as antigen presenting cells (APCs), in Stimulation Medium (Prepare in DMEM + Pen/Strep/L-Glut + NEAA + RPMI Vitamins + 2ME + Rat Serum + Ovalbumin, at the final concentrations noted in the materials list.)
Rat serum preparation
<ol>
<li>We take serum from the rats we sacrifice as thymus donors.</li>
<li>Rats are deeply anesthetized by halothane inhalation and as much blood as possible is drawn by cardiac perfusion (5-10 ml per rat) using 10 ml syringes and 23 G 1" needles, after which the rats are immediately decapitated to ensure death.</li>
<li>The blood is allowed to clot for 10 min at 37&deg;C followed by 10 min on ice.</li>
<li>The clot is removed and the remaining liquid spun.</li>
<li>The serum is collected, sterile-filtered and stored at -20&deg;C.</li>
</ol>
Preparation of the antigen presenting cells
<ol>
<li>Take the thymus of a decapitated rat using sterile instruments and place it in PBS containing penicillin and streptomycin (PBS-PS), on ice.</li>
<li>Take the thymus to a tissue culture hood, clean it from blood clots and other contaminating tissue and cut it in small pieces into a cell strainer (Fisher # 08-771-2) placed in a 10 cm petri dish containing 10 ml of PBS-PS.</li>
<li>Each piece is pressed through the cell strainer using the back of a sterile 1 ml syringe plunger. Collect the single cell suspension into a 50 ml tube on ice.</li>
<li>Rinse the cell strainer with 20-40 more ml of PBS-PS. Centrifuge the suspension at 1250g and resuspend the pellet in PBS-PS, 5 ml/thymus, on ice.</li>
<li>Irradiate this suspension (30 Gy) in a gamma irradiator and immediately wash it twice with 50 ml PBS-PS. Note: The best thymus donors are young adult rats, 5-7 weeks old. Rats up to 10 weeks may be used but the number of cells decreases with time. Gender is not important although some people say that males are better thymus donors. We always use females because all our animals are housed in the same room and we don't want males and females in the same room. </li>
</ol>
Stimulation
<ol>
<li>Wash the T lymphocytes once and count them.</li>
<li>Count the antigen-presenting cells.</li>
<li>Mix 3 x 106 ovalbumin-specific T cells with 150 x 106 APCs in a 10 cm petri dish containing 10 ml of stimulation medium.</li>
<li>Incubate for 48 hours at 37&deg;C.</li>
</ol>
2. Adoptive transfer of the ovalbumin-specific T lymphocytes
<ol>
<li>The rats we use as recipients are females 7-10 weeks old.</li>
<li>Count the T lymphocytes, they should be large and round. The antigen-presenting cells will be dead.</li>
<li>Centrifuge the T lymphocytes and resuspend them at 10 x 106 cells/ml in PBS.</li>
<li>Inject 1 ml intraperitoneally using 3 ml syringes and 23 G 1" needles.</li>
</ol>
3. Challenge with antigen in the ear
<ol>
<li>Two days after adoptive transfer, inject 20 &mu;l of a 1 mg/ml ovalbumin solution (= relevant antigen) in one ear and saline (or irrelevant antigen) in the other ear. Do NOT filter the antigen solution as you may lose aggregates that enhance the inflammatory reaction. Note: The rats should be anesthetized to avoid movements. If you are right-handed, hold the ear between your left thumb and index finger, the index finger being inside the ear and the rat facing you. </li>
<li>Inject the solution in the pinna of the ear using 27G &frac12;" needles. Try to inject all the rats exactly at the same place.</li>
</ol>
4. Measurement of DTH
<ol>
<li>The ear will start turning red and swell as a sign of inflammation. Measure the thickness of each ear with a spring-loaded micrometer (for example model PK-0505 from Mitutoyo).</li>
<li>Take 5-6 measurements for each ear at each time-point and calculate the average.</li>
<li>The maximum swelling should occur between 24 and 72 hours.</li>
</ol>

<ol>
	<li>Beeton, C., Barbaria, J., Devaux, J., Benoliel, A. -. M., Gola, M., Sabatier, J. -. M., Bernard, D., Crest, M., Beraud, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+blocking+of+voltage-gated+K++channels+treats+experimental+autoimmune+encephalomyelitis+and+inhibits+T-cell+activation.">Selective blocking of voltage-gated K+ channels treats experimental autoimmune encephalomyelitis and inhibits T-cell activation.</a> J. Immunol. , 166-936 (2001).</li><li>Beeton, C., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+monitoring+of+active+delayed+type+hypersensitivity+(DTH)in+rats.">Induction and monitoring of active delayed type hypersensitivity (DTH)in rats.</a> J Vis Exp. (5), (2007).</li></ol>

The antigenic challenge can also be performed at other sites, for example in the skin of the back (2). We however find that challenging. Using the pinna of the ear, as shown in this video, allows for the most precise measurement of the DTH reaction.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco/Invitrogen</td>
<td>12430-062</td>
<td>500ml</td>
</tr>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8636</td>
<td>1 mM final, in Stimulation medium</td>
</tr>
<tr>
<td>Penicillin &#x2013; Streptomycin &#x2013; L-Glutamine</td>
<td>&nbsp;</td>
<td>Cambrex/BioWhittaker</td>
<td>17-718R</td>
<td>100 u/ml - 100 &micro;g/ml - 4 mM respectively, final, in Stimulation Medium </td>
</tr>
<tr>
<td>Non essential amino acids</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R7131</td>
<td>NEAA, 1% final, in Stimulation Medium</td>
</tr>
<tr>
<td>RPMI vitamins</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R7256</td>
<td>1% final, in Stimulation medium</td>
</tr>
<tr>
<td>beta-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M6250</td>
<td>2ME, 50 &micro;M final, in Stimulation Medium</td>
</tr>
<tr>
<td>Syngeneic rat serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1% final. See explanation in Protocol.</td>
</tr>
<tr>
<td>Ovalbumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5503</td>
<td>10 &micro;g/ml final, in Stimulation Medium.</td>
</tr>		</tbody>
		</table>
		</div>

induction and monitoring of adoptive delayed-type hypersensitivity in rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Watch this Scientific Journal Video about Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats at JoVE.com

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

induction-monitoring-adoptive-delayed-type-hypersensitivity

Universidad de Cartagena UDEC

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JoVE Journal

Biology

11.9K Views.  University of California, Irvine (UCI). Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

Video: Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin (KLH).
Here we demonstrate how to induce an active DTH reaction in Lewis rats. We will first prepare a water-in-oil emulsion of KLH, our antigen of interest, in complete Freund's adjuvant and inject this emulsion subcutaneously to rats. This will prime the immune system to develop memory T cells directed to KLH. Seven days later we will challenge the rats intradermally on the back with KLH on one side and with ovalbumin, an irrelevant antigen, on the other side. The inflammatory reaction will be visible 16-72 hours later and the red and swollen area will be measured as an indication of DTH severity.

Induction and Monitoring of Active Delayed Type Hypersensitivity DTH in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes. Here we demonstrate how to induce active DTH in Lewis rats and monitor the inflammatory response.

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7- effector / memory T lymphocytes. Here, we demonstrate a method for inducing and recording the progress of a DTH reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the CCR7- effector / memory T cell response.
An adoptive DTH is induced by the intraperitoneal injection of GFP-labeled Ova-specific CCR7- effector / memory T cell line (Beeton, C J. Visualized Experiments, Issue 8). Cells are then allowed to equilibrate in the rat for 48 hours before challenge by injecting one ear with saline (control ear) and the other with a 1:1 mix of Ova and Ova conjugated to Texas-Red (Ova-TR) to allow visualization of resident antigen-presenting cells.
We describe a method of tissue preparation useful for imaging the motility of cells within the deep dermal layer during an immune response, in conjunction with visualization of collagen fibers by second harmonic generation. Ear tissue is cut into 5 x 5 mm squares (slightly larger is better) and mounted onto plastic cover slips using Vetbond&trade;, which are then secured using silicone grease in an imaging chamber and superfused by oxygen-bubbled tissue culture medium at 37&deg;C.

Here we demonstrate a method for inducing and recording the progress of a delayed type-hypersensitivity (DTH) reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the effector / memory T cell response.

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7- effector / memory T lymphocytes. Here, we demonstrate a ...

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Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

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Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Summary

Explore More Videos

Chapters in this video

Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Assay for Neural Induction in the Chick Embryo

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Induction and Testing of Hypoxia in Cell Culture

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Induction and Analysis of Epithelial to Mesenchymal Transition

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells