Name: Examination of Thymic Positive and Negative Selection by Flow Cytometry (Video) | JoVE Journal
Uploaded: 2012-10-08T13:00:00.000+00:00
Duration: 14 min 29 s
Description: Learn flow cytometry methods to examine T cell development in genetically manipulated mice. This technique identifies lymphocyte populations and reveals critical genes for immune tolerance and repertoire generation.

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5 minutes

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 the fur is matted down.</li>
 <li>Using surgical scissors, begin dissection by making a ventral incision in the abdominal skin just above the genitalia. Extend the incision upwards to the chin. </li>
 <li>From the midline, extend the incision down along all limbs. Pull back the loose skin and pin it down to the dissection surface.</li>
 <li>Harvest the thymus:
 <ol>
 <li>Lift the bottom tip of the sternum to make an incision.</li>
 <li>Avoiding the liver, cut the diaphragm to detach the rib cage and then cut the rib cage to each side. Cut the ribcage upwards on each side, taking care to avoid the lungs and heart.</li>
 <li>Gently pull the ribcage back with forceps. The thymus is a white, bilobed organ located above the heart. Using the flat edge of the forceps to grasp the bottom of the lobes, gently pull the thymus and place it on the mesh screen.</li>
 </ol>
 </li>
 <li>Harvest the spleen:
 <ol>
 <li>The spleen is a red, surfboard-shaped organ located on the left side of the mouse's abdominal cavity, below the liver.</li>
 <li>Make an incision into the abdominal cavity. Gently pull out the spleen with one pair of forceps, using the second pair to tease away connective tissue. Place the spleen on a separate mesh screen.</li>
 </ol>
 </li>
</ol>
2. Cell Preparation
<ol>
 <li>Using a plunger from a 3 ml syringe, grind organs into the mesh screen until only connective tissue and fat remains. Rinse mesh with HBSS from the dish three times. Use a new plunger for each tissue sample.
 <ol>
 <li>Other options for homogenizing tissue can be used in place of this method.</li>
 </ol>
 </li>
 <li>Count cells using a hemocytometer.</li>
 <li>Pellet cells by centrifugation at 335 x g for 5 min at 4 &deg;C.</li>
 <li>Resuspend splenocytes in 500 &mu;l of ACK lysis buffer (0.15 M NH4Cl, 10 mM KHC03, 0.1 mM Na2EDTA, pH 7.2) for 10 min at room temperature. Resuspend thymocytes at 20 x 106 cells/ml in FACS buffer (PBS, 1% FCS, 0.02% sodium azide) and set aside on ice.</li>
 <li>Return splenocytes to isotonicity by adding 5 ml of HBSS. Pellet splenocytes by centrifugation and resuspend at 20 x 106 cells/ml in FACS buffer. If cells need to remain sterile, you may use sterile RPMI + 10% FCS.</li>
</ol>
3. Staining Cells for Flow Cytometry
The purpose of this protocol is to outline strategies for the analysis of positive and negative selection using wildtype (WT) and HYcd4 mice. For general considerations regarding flow cytometry experimental design, acquisition, and analysis, we refer readers to an excellent review by Tung et al.11
<ol>
 <li>Aliquot 4 x 106 thymocytes per flow cytometry sample to each well of a 96-well plate, as well as 1 x 106 WT splenocytes per compensation control. If using transgenic mice, you should include a WT mouse as a control in your experiment.</li>
 <li>Block Fc receptors by incubating cells with anti-CD16/32 (clone 2.4G2) for 10 min on ice.</li>
 <li>Spin plate at 335 x g at 4 &deg;C for 5 min. Remove lid and dispel liquid from wells by flicking the plate once, face down into a sink. Resuspend each well in 200 &mu;l of FACS buffer. Repeat the wash.</li>
 <li>Prepare antibody cocktails as listed below, using the optimal concentration of each antibody based on dilution in 200 &mu;l of FACS buffer per well. The optimal concentration of each antibody is defined as the lowest concentration needed to give the largest separation of positive and negative populations. If there is no negative population for a given antibody, an isotype antibody should be included. The total volume per well can be scaled down (e.g. to 100 &mu;l per well) if desired.
 <ol>
 <li>WT thymus: anti-TCR&beta;, anti-CD4, anti-CD8, anti-CD69 or anti-CD5, anti-CD24</li>
 <li>HYcd4 thymus: anti-HY TCR (T3.70), anti-CD4, anti-CD8, anti-CD69 or anti- CD5, anti-CD24 
To obtain better separation of CD69lo and CD69hi populations in thymocytes, it is recommended that a biotinylated anti-CD69 primary antibody be used, followed by a secondary stain containing fluorochrome-conjugated streptavidin. We use the same strategy for CD5 staining.
 </li>
 </ol>
 </li>
 <li>Incubate cells with 200 &mu;l of antibody cocktail in FACS buffer for 30 min on ice in the dark.</li>
 <li>Concurrent with antibody cocktail staining, stain each compensation control with a single fluorochrome-conjugated antibody. Ideally, compensation staining involves the same antibodies as used in the antibody cocktail. However, staining for each individual antigen may not be feasible for larger experiments when many antigens are being assayed. Therefore, staining for either anti-CD4 or anti-CD8 is recommended due to high expression of these antigens, or the use of compensation beads. Stain in FACS buffer for 30 min on ice in the dark. Leave one compensation control unstained. </li>
 <li>Wash cells twice with FACS buffer.</li>
 <li>Resuspend cells in FACS buffer and transfer to FACS tubes.</li>
 <li>Acquire samples on flow cytometer. Include acquisition of FSC-A and FSC-W to allow for doublet discrimination.</li>
</ol>
4. Analysis of Flow Cytometry Data - Non-TCR Transgenic Mice
We use FlowJo for flow cytometry data analysis. Refer to Figure 2A for the gating strategy for non-TCR transgenic mice.
<ol>
 <li>Using FSC-A by SSC, electronically gate on the "lymphocyte" population.</li>
 <li>Within the "lymphocyte" population, use FSC-A by FSC-W to electronically gate the FSC-Wlo population to exclude cell doublets and aggregrates. This is the "singlet" gate.</li>
 <li>Using the events in the "singlet" gate, plot CD8 by CD4. Draw gates for DN, DPdull, DPbright, CD4+CD8lo, CD4SP and CD8SP populations as depicted in Figure 2B.</li>
 <li>Under the CD4SP and CD8SP gates, create TCR&beta; by CD24 plots. Use the quadrant gating tool to draw gates as depicted in Figure 2C.</li>
 <li>Create a new plot from the "singlet" gate: TCR&beta; by CD69. Draw gates A, B, C, D as depicted in Figure 2D.</li>
 <li>Create another plot from the "singlet" gate: TCR&beta; by CD5. Draw gates i, ii, iii, iv as depicted in Figure 2E. This is another strategy for examining positive selection.</li>
 <li>Apply DN, DPdull, DPbright, CD4int, CD4SP and CD8SP gates from under "singlet" to gates i, ii, iii, iv, A, B, C, D (Figure 2D, E).</li>
 <li>Calculate the number of cells in a particular subset by multiplying organ cellularity by the frequency of each subsequent gate until you reach your target population. For example, the number of TCR&beta;hiCD69- CD8SP thymocytes would be determined by thymus cellularity x %TCRbhi CD69- (fraction D) x %CD8SP.
 <ol>
 <li>Counting already takes into account the live and dead cells so do not include the "lymphocyte" gate in your calculations.</li>
 </ol>
 </li>
</ol>
5. Analysis of Flow Cytometry Data - TCR Transgenic Mice
Refer to Figure 3A for the gating strategy for TCR transgenic mice. 
<ol>
 <li>Follow steps 4.1 and 4.2 as in the previous section.</li>
 <li>Under the "singlet" gate, create a T3.70 by SSC plot and gate on the T3.70+ cell population to analyze antigen-specific T cells (Figure 3B). In some circumstances, it may be of interest to compare this population to the non-antigen specific (T3.70-) T cell population.</li>
 <li>Within the T3.70+ population, draw DN, DP, CD4SP and CD8SP gates (Figure 3C).
 <ol>
 <li>For WT control samples, draw these gates under the &#x201C;singlet&#x201D; gate or create a T3.70- gate as there will be very few T3.70+ cells.</li>
 </ol>
 </li>
 <li> Under the CD8SP gate, create a T3.70 by CD24 plot. Use the quadrant gating tool to divide cells into four populations (Figure 3F).</li>
 <li>Create an overlaid histogram depicting CD69 expression in the DP compartment of WT mice and T3.70+ DP compartment of HYcd4 mice (Figure 4A). Repeat to examine CD5 expression (Figure 4B).</li>
 <li>Calculate the absolute number of cells in each subset of interest as in 4.8.</li>
</ol>

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</ol>

No conflicts of interest declared.

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><tbody><tr><td>HyClone Hank&#039;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&amp;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&amp;#946; </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 colspan="4">&lt;p class=&quot;jove_content&quot;&gt;*XX varies by fluorochrome and YY varies by vial size.&lt;/p&gt;</td></tr></tbody></table>

examination of thymic positive and negative selection by flow cytometry

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...

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 ...

The overall goal of the following experiment is to examine thy development in mice by flow cytometry. This is achieved by preparing single cell suspensions from the mouse, thymus and spleen. As a second step, the thymocytes and cytes are stained with a cocktail of antibodies for the identification of specific thy populations.Ultimately, the frequency and number of various lymphocyte populations can be evaluated by flow cytometric analysis. This method can help answer key questions in T cell development, such as which genes and pathways are important for generating a functional yet cell tolerant T cell repertoire. Though this method can provide insight into T-cell development in the thymus, it can also be applied in the examination of the development of other immune cells.Generally, individuals new to this method will struggle in the design of their antibody cocktails for flow cytometry. Before starting the dissection, place a sterile steel mesh screen into a 60 by 15 millimeter Petri dish. Then add five milliliters of HBSS to the dish and store the dish on ice to harvest the thymus.First, use a pair of forceps to lift the bottom tip of the sternum and reveal the diaphragm. Next, avoiding the liver, cut the diaphragm to detach the ribcage and then cut the ribcage in an upwards direction on each side. Taking care to avoid the lungs and heart.Now use the forceps to gently pull the rib cage back. The thymus is a white bi lobed organ located above the heart. Use the flat edge of the forceps to grasp the bottom of the lobes.Then gently pull the thymus out and place it onto one of the previously prepared mesh screens. To harvest the spleen, make an incision through the peritoneum to expose the abdominal cavity. The spleen is a red surfboard shaped organ located on the left side of the mouse's abdominal cavity below the liver.Then use one pair of forceps to pull out the spleen and another to tease away the connective tissue. Then place the dissected spleen on a separate mesh screen. Now use the plunger from a three milliliter syringe to grind each organ into its mesh screen until only connective tissue and fat remain pipette the cells and HBSS into a 15 milliliter conical tube.Then rinse each mesh with fresh HBS S3 times. Next, pellet the cells for five minutes at 335 times G and four degrees Celsius. After aspirating the supernatant resus, suspend the cytes in 500 microliters of a CK lysis buffer for 10 minutes at room temperature while the splenic red blood cells are being lyed Resus.Suspend the thymocytes at 20 times 10 to the six cells per milliliter in fax buffer, and set them aside on ice. Now add five milliliters of HBSS to return the cytes to isotonicity after spinning down the cells again, resus suspend the RBC free pellet at 20 times 10 to the sixth cells per milliliter in fax buffer. Begin this step by Ali Watting.Four times 10 to the sixth of the reserved THYMOCYTES per flow cytometry sample to each well of a 96 well plate. Next, add cytes to the wells of a 96 well plate for compensation controls. Then block the FC receptors in each of the cell samples by incubation with anti CD 1632 for 10 minutes on ice.Now spin down the plate and then dispel the liquid from the wells by flicking the plate once face down into a sink, then resus suspend each well in 200 microliters of fax buffer and repeat the wash. Next, incubate the experimental samples with 200 microliters of antibody cocktail or single fluorochrome conjugated antibodies for the compensation controls. All in fax buffer on ice in the dark.After 30 minutes, wash the cells twice with fax buffer and then after resus suspending the cells in fax buffer transfer the samples to fax tubes in physiological TCR transgenic models and wild type mice. Positive selection begins at the double positive bright stage before moving into the double positive dull stage. After antigen encounter the double positive dull thymocytes, then enter a transitional CD four positive CD eight low stage before becoming CD four.Single positive or CD eight single positive thymocytes mature single positive thymocytes are characterized by their high TCR expression and loss of CD 24. While the CD eight by CD four profile can reveal defects in positive selection, examining TCR beta by CD 69 or CD five can provide further insight into where the defect lies. Both CD 69 and CD five are upregulated after TCR stimulation with stronger interactions driving higher expression of these markers.In this TCR beta by CD 69 plot in a wild type mouse, the TCR R beta low CD 69 negative gait represents a population of pre-selection double positive thymocytes. Whereas this TCR beta intermediate CD 69 positive gait represents a transitional population directly after TCR engagement and consists of double positive bright with some double positive dull and CD four positive CD eight low cells. Here the TCR R beta high CD 69 positive gait illustrates the population of cells directly post positive selection, which consist of double positive dull CD four positive, CD eight low and CD four single positive cells.Finally, this TCR beta high CD 69 negative gait shows a more mature population of cells that primarily consists of CD four and CD eight single positive cells. The absence of the TCR R beta intermediate CD 69 positive and TCR R beta high CD 69 positive populations may be indicative of impaired positive selection changes in the ratio of the CD four single positive to CD eight. Single positive cells within the TCR beta high CD 69 negative population may suggest alterations in lineage commitment.The loss of the TCR beta high CD 69 positive and TCR beta high CD 69 negative populations may reflect survival issues following positive selection. Examining TCR beta by CD five is another strategy to identify pre and post positive selection populations. These first two populations consist primarily of double positive bright thymocytes.The TCR beta low CD five low population represents preselection double positive thymocytes, and the TCR beta intermediate CD five intermediate population is made up of the cells that initiate positive selection. Impaired generation of this or the previous population suggests defective positive selection. The TCR R beta intermediate CD five high population, however, represents thymocytes in the process of undergoing positive selection and consists primarily of double positive dull and CD four positive.CD eight low thymocytes. The TCR beta high CD five high population consists primarily of post positive selection. Single positive thymocytes changes in the ratio of CD four, single positive to CD eight.Single positive cells within this population, despite normal preceding populations may suggest alterations in lineage commitment. Further, an absence of this population may indicate a decreased survival of thymocytes following positive selection as negative selection involves the deletion of small antigen specific populations. Defects in this process are best observed using TCR transgenic mice in HY CD four mice thymocytes expressing the transgenic H-Y-T-C-R can be detected with the monoclonal antibody T 3.7.The H-Y-T-C-R recognizes the male specific HY antigen presented within MHC Class one db. Thus HY CD four male mice undergo negative selection of H-Y-T-C-R positive thymocytes as indicated by a reduction in T three point 70 positive double positive THYMOCYTES numbers, and a more dramatic decrease in T 3.7 positive CD eight, single positive thy cyte numbers. In contrast, HY CD four female mice undergo positive selection to generate T 3.7 positive CD eight single positive T cells.While a reduction in double positive thy numbers is indicative of negative selection, the absence of antigen specific single positive thymocytes is the most accurate measure. Further examination of the few T three point 70 positive CD eight single positive thymocytes in HY CD four male mice reveals that most are CD 24 high immature cells. While most T three point 70 positive CD eight single positive thymocytes in the HY CD four female have reached maturity and are CD 24 low, providing further support, that negative selection occurs in HY CD four male mice.One caveat of the TCR transgenic models is that it is difficult to further characterize positive selection by identifying populations based on TCR and CD 69 or CD five expression due to high TCR expression throughout thy CYTE development, HY CD four female mice have a large population of T three point 70 positive double positive thymocytes that undergo positive selection. As indicated by an increase in the CD 69 positive population compared to wild type negative selection involves a higher affinity TCR stimulus than positive selection. This is indicated by higher expression of CD 69 on T three point 70 positive double positive thymocytes in HY CD four male mice resulting in a shift of the histogram peak to the right.Similar trends are seen with CD five expression when analyzing thymic selection in genetically manipulated mice. Examining CD 69 or CD five expression can determine whether the defect lies with the TCR signaling or a downstream outcome of TCR stimulation. Once mastered, this technique can be performed in two and a half hours for cell preparation and staining, plus an additional one hour for data collection on the flow cytometer.If performed correctly, each additional mouse will add approximately 30 minutes. While attempting this procedure, it is important to remember to handle the thymocytes gently and to ensure you have planned the staining strategy beforehand. Following this procedure.Other assays such as killing assays or cytokine production assays can be performed in order to answer additional questions like whether the exported thymocytes function properly. After watching this video, you should have a good understanding of how to analyze thymic site development using flow cytometry.

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