The authors would like to thank Bing Zhang for his technical assistance. This work was funded by the Canadian Institutes for Health Research (MOP-86595). T.A.B is a CIHR New Investigator and AHFMR Scholar. Q.H. is supported by a CIHR Canada Graduate Scholarship - Doctoral and an AIHS Full-time Studentship. S.A.N. is supported by a Queen Elizabeth II Graduate Scholarship. A.Y.W.S. is supported by an NSERC Postgraduate Scholarship - Doctoral.

Refer to Figure 1 for an overall scheme of the experimental protocol.
1. Dissection
<ol>
 <li>Place sterile steel mesh screen into 60 x 15 mm Petri dish. One unit is needed per tissue sample.</li>
 <li>Add 5 ml of Hank's balanced salt solution (HBSS) to each dish. Keep dishes on ice.</li>
 <li>Euthanize mice with CO2.</li>
 <li>Secure mouse to dissection surface, ventral side facing up. Spray mouse with 70% ethanol for sterilization and to ensure that t...

<ol>
	<li>Liston, A., Lesage, S., Wilson, J., Peltonen, L., Goodnow, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aire+regulates+negative+selection+of+organ-specific+T+cells.">Aire regulates negative selection of organ-specific T cells.</a> Nat. Immunol. 4, 350-354 (2003).</li><li>Liston, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+dosage--limiting+role+of+Aire+in+thymic+expression,+clonal+deletion,+and+organ-specific+autoimmunity.">Gene dosage--limiting role of Aire in thymic expression, clonal deletion, and organ-specific autoimmunity.</a> J. Exp. Med. 200, 1015-1026 (2004).</li><li>Hogquist, K. A., Baldwin, T. A., Jameson, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymocytes+between+the+beta-selection+and+positive+selection+checkpoints+are+nonresponsive+to+IL-7+as+assessed+by+STAT-5+phosphorylation.">Thymocytes between the beta-selection and positive selection checkpoints are nonresponsive to IL-7 as assessed by STAT-5 phosphorylation.</a> J. Immunol. 172, 4235-4244 (2004).</li><li>Ueno, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR7+signals+are+essential+for+cortex-medulla+migration+of+developing+thymocytes.">CCR7 signals are essential for cortex-medulla migration of developing thymocytes.</a> J. Exp. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projection+of+an+immunological+self+shadow+within+the+thymus+by+the+aire+protein.">Projection of an immunological self shadow within the thymus by the aire protein.</a> Science. 298, 1395-1401 (2002).</li><li>Kurts, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+class+I-restricted+exogenous+presentation+of+self+antigens+in+vivo.">Constitutive class I-restricted exogenous presentation of self antigens in vivo.</a> J. Exp. Med. 184, 923-930 (1996).</li><li>Nitta, T., Nitta, S., Lei, Y., Lipp, M., 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=CCR7-mediated+migration+of+developing+thymocytes+to+the+medulla+is+essential+for+negative+selection+to+tissue-restricted+antigens.">CCR7-mediated migration of developing thymocytes to the medulla is essential for negative selection to tissue-restricted antigens.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 17129-17133 (2009).</li><li>Bouneaud, C., Kourilsky, P., Bousso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+negative+selection+on+the+T+cell+repertoire+reactive+to+a+self-peptide:+a+large+fraction+of+T+cell+clones+escapes+clonal+deletion.">Impact of negative selection on the T cell repertoire reactive to a self-peptide: a large fraction of T cell clones escapes clonal deletion.</a> Immunity. 13, 829-840 (2000).</li><li>Gallegos, A. 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The protocol presented here can be used to examine positive and negative selection in non-TCR transgenic and TCR transgenic mice. This protocol describes the staining of surface antigens. For further analysis of molecular mechanisms, it is often necessary to perform intracellular staining. We use the BD Biosciences Cytofix/Cytoperm Kit for most intracellular proteins and the BD Biosciences Foxp3 Staining Kit for transcription factors. We usually acquire our samples immediately after staining. However, samples can ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>HyClone Hank's balanced salt solution</td>
 <td>Thermo Scientific</td>
 <td>SH30030.02</td>
 <td></td>
 </tr>
 <tr>
 <td>Metal mesh screens</td>
 <td>Cedarlane</td>
 <td>CX-0080-E-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Petri dishes (60 x 15 mm)</td>
 <td>Fisher Scientific</td>
 <td>877221</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringes (3 ml)</td>
 <td>BD Biosciences</td>
 <td>309657</td>
 <td></td>
 </tr>
 <tr>
 <td>Conical tubes (15 ml)</td>
 <td>Sarstedt</td>
 <td>62.554.205</td>
 <td></td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td>Zeiss - Primo Star</td>
 <td>415500-00XX-000</td>
 <td></td>
 </tr>
 <tr>
 <td>Hemocytometer </td>
 <td>Hausser Scientific</td>
 <td>3110</td>
 <td></td>
 </tr>
 <tr>
 <td>96-well plate</td>
 <td>Sarstedt</td>
 <td>82.1582.001</td>
 <td></td>
 </tr>
 <tr>
 <td>Multichannel pipette</td>
 <td>Fisherbrand</td>
 <td>21-377-829</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal calf serum</td>
 <td>PAA</td>
 <td>A15-701</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate buffered saline</td>
 <td>Fisher Scientific</td>
 <td>SH3025802</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium azide</td>
 <td>IT Baker Chemical Co.</td>
 <td>V015-05</td>
 <td></td>
 </tr>
 <tr>
 <td>FcR blocking reagent</td>
 <td></td>
 <td></td>
 <td>Clone 2.4G2</td>
 </tr>
 <tr>
 <td>Anti-mouse HY TCR </td>
 <td>eBioscience</td>
 <td>XX-9930-YY*</td>
 <td>Clone T3.70</td>
 </tr>
 <tr>
 <td>Anti-mouse CD4</td>
 <td>eBioscience</td>
 <td>XX-0042-YY*</td>
 <td>Clone RM4-5</td>
 </tr>
 <tr>
 <td>Anti-mouse CD8&alpha; </td>
 <td>eBioscience</td>
 <td>XX-0081-YY*</td>
 <td>Clone 53-6.7</td>
 </tr>
 <tr>
 <td>Anti-mouse CD24</td>
 <td>eBioscience</td>
 <td>XX-0242-YY*</td>
 <td>Clone M1/69</td>
 </tr>
 <tr>
 <td>Anti-mouse TCR&beta; </td>
 <td>eBioscience</td>
 <td>XX-5961-YY*</td>
 <td>Clone H57-597</td>
 </tr>
 <tr>
 <td>Anti-mouse CD69 Biotinylated</td>
 <td>eBioscience</td>
 <td>13-0691-YY*</td>
 <td>Clone H1.2F3</td>
 </tr>
 <tr>
 <td>Anti-mouse CD5 Biotinylated</td>
 <td>eBioscience</td>
 <td>13-0051-YY*</td>
 <td>Clone 53-7.3</td>
 </tr>
 <tr>
 <td>Streptavidin</td>
 <td>eBioscience</td>
 <td>XX-4217-YY*</td>
 <td></td>
 </tr>
 <tr>
 <td>Flow cytometer</td>
 <td>BD Biosciences - FACS Canto </td>
 <td>338962</td>
 <td></td>
 </tr>
 <tr>
 <td>FACS tubes</td>
 <td>BD Biosciences</td>
 <td>352052</td>
 <td></td>
 </tr>
 <tr>
 <td>Flow cytometry analysis software</td>
 <td>TreeStar - Flowjo</td>
 <td>FlowJo v7/9</td>
 <td></td>
 </tr>
 <tr>
 <td>HyClone RPMI - 1640 medium</td>
 <td>Thermo Scientific</td>
 <td>SH30027.01</td>
 <td></td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>*XX varies by fluorochrome and YY varies by vial size.</td>
 </tr>
 </tbody>
</table>

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.

In physiological TCR transgenic models and WT mice, positive selection begins at the DPbright stage before moving into the DPdull stage after antigen encounter. DPdull thymocytes then enter a transitional CD4+CD8lo stage before becoming CD4SP or CD8SP thymocytes (Figure 2B). Mature SP thymocytes are characterized by high TCR expression and loss of CD24 (Figure 2C). While the CD8 by CD4 profile can reveal defects in positive se...

Watch this Scientific Journal Video about Examination of Thymic Positive and Negative Selection by Flow Cytometry at JoVE.com

Examination of Thymic Positive and Negative Selection by Flow Cytometry

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.

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

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Immunology and Infection

21.6K visualizações.  University of Alberta. Apresenta-se um método de citometria de fluxo com base para examinar o desenvolvimento das células T In vivo Usando camundongos geneticamente manipulados em um tipo selvagem ou de fundo do receptor de células T transgênico.