Examination of Thymic Positive and Negative Selection by Flow Cytometry

Summary

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.

Abstract

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) α and β 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 disorders^1-4.

T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the αβTCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the αβTCR 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 thymocytes^7-9. 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 TCRα chain at the DN stage, resulting in premature negative selection. Our lab has developed the HY^cd4 model, in which the transgenic HY TCRα 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 HY^cd4 mouse model. While negative selection in HY^cd4 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.

Protocol

Refer to Figure 1 for an overall scheme of the experimental protocol.

1. Dissection

1. Place sterile steel mesh screen into 60 x 15 mm Petri dish. One unit is needed per tissue sample.
2. Add 5 ml of Hank's balanced salt solution (HBSS) to each dish. Keep dishes on ice.
3. Euthanize mice with CO₂.
4. 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.
5. 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.
6. From the midline, extend the incision down along all limbs. Pull back the loose skin and pin it down to the dissection surface.
7. Harvest the thymus:
   1. Lift the bottom tip of the sternum to make an incision.
   2. 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.
   3. 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.
8. Harvest the spleen:
   1. The spleen is a red, surfboard-shaped organ located on the left side of the mouse's abdominal cavity, below the liver.
   2. 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.

2. Cell Preparation

1. 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.
   1. Other options for homogenizing tissue can be used in place of this method.
2. Count cells using a hemocytometer.
3. Pellet cells by centrifugation at 335 x g for 5 min at 4 °C.
4. Resuspend splenocytes in 500 μl 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.
5. 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.

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 HY^cd4 mice. For general considerations regarding flow cytometry experimental design, acquisition, and analysis, we refer readers to an excellent review by Tung et al.¹¹

1. 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.
2. Block Fc receptors by incubating cells with anti-CD16/32 (clone 2.4G2) for 10 min on ice.
3. Spin plate at 335 x g at 4 °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 μl of FACS buffer. Repeat the wash.
4. Prepare antibody cocktails as listed below, using the optimal concentration of each antibody based on dilution in 200 μl 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 μl per well) if desired.
   1. WT thymus: anti-TCRβ, anti-CD4, anti-CD8, anti-CD69 or anti-CD5, anti-CD24
   2. HY^cd4 thymus: anti-HY TCR (T3.70), anti-CD4, anti-CD8, anti-CD69 or anti- CD5, anti-CD24
      To obtain better separation of CD69^lo and CD69^hi 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.
5. Incubate cells with 200 μl of antibody cocktail in FACS buffer for 30 min on ice in the dark.
6. 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.
7. Wash cells twice with FACS buffer.
8. Resuspend cells in FACS buffer and transfer to FACS tubes.
9. Acquire samples on flow cytometer. Include acquisition of FSC-A and FSC-W to allow for doublet discrimination.

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.

1. Using FSC-A by SSC, electronically gate on the "lymphocyte" population.
2. Within the "lymphocyte" population, use FSC-A by FSC-W to electronically gate the FSC-W^lo population to exclude cell doublets and aggregrates. This is the "singlet" gate.
3. Using the events in the "singlet" gate, plot CD8 by CD4. Draw gates for DN, DP^dull, DP^bright, CD4⁺CD8^lo, CD4SP and CD8SP populations as depicted in Figure 2B.
4. Under the CD4SP and CD8SP gates, create TCRβ by CD24 plots. Use the quadrant gating tool to draw gates as depicted in Figure 2C.
5. Create a new plot from the "singlet" gate: TCRβ by CD69. Draw gates A, B, C, D as depicted in Figure 2D.
6. Create another plot from the "singlet" gate: TCRβ by CD5. Draw gates i, ii, iii, iv as depicted in Figure 2E. This is another strategy for examining positive selection.
7. Apply DN, DP^dull, DP^bright, CD4^int, CD4SP and CD8SP gates from under "singlet" to gates i, ii, iii, iv, A, B, C, D (Figure 2D, E).
8. 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 TCRβ^hiCD69- CD8SP thymocytes would be determined by thymus cellularity x %TCRbhi CD69- (fraction D) x %CD8SP.
   1. Counting already takes into account the live and dead cells so do not include the "lymphocyte" gate in your calculations.

5. Analysis of Flow Cytometry Data - TCR Transgenic Mice

Refer to Figure 3A for the gating strategy for TCR transgenic mice.

1. Follow steps 4.1 and 4.2 as in the previous section.
2. 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.
3. Within the T3.70⁺ population, draw DN, DP, CD4SP and CD8SP gates (Figure 3C).
   1. For WT control samples, draw these gates under the “singlet” gate or create a T3.70- gate as there will be very few T3.70⁺ cells.
4. Under the CD8SP gate, create a T3.70 by CD24 plot. Use the quadrant gating tool to divide cells into four populations (Figure 3F).
5. Create an overlaid histogram depicting CD69 expression in the DP compartment of WT mice and T3.70⁺ DP compartment of HY^cd4 mice (Figure 4A). Repeat to examine CD5 expression (Figure 4B).
6. Calculate the absolute number of cells in each subset of interest as in 4.8.

Results

In physiological TCR transgenic models and WT mice, positive selection begins at the DP^bright stage before moving into the DP^dull stage after antigen encounter. DP^dull thymocytes then enter a transitional CD4⁺CD8^lo 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...

Discussion

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 ...

Materials

Name	Company	Catalog Number	Comments
HyClone Hank's balanced salt solution	Thermo Scientific	SH30030.02
Metal mesh screens	Cedarlane	CX-0080-E-01
Petri dishes (60 x 15 mm)	Fisher Scientific	877221
Syringes (3 ml)	BD Biosciences	309657
Conical tubes (15 ml)	Sarstedt	62.554.205
Microscope	Zeiss - Primo Star	415500-00XX-000
Hemocytometer	Hausser Scientific	3110
96-well plate	Sarstedt	82.1582.001
Multichannel pipette	Fisherbrand	21-377-829
Fetal calf serum	PAA	A15-701
Phosphate buffered saline	Fisher Scientific	SH3025802
Sodium azide	IT Baker Chemical Co.	V015-05
FcR blocking reagent			Clone 2.4G2
Anti-mouse HY TCR	eBioscience	XX-9930-YY*	Clone T3.70
Anti-mouse CD4	eBioscience	XX-0042-YY*	Clone RM4-5
Anti-mouse CD8α	eBioscience	XX-0081-YY*	Clone 53-6.7
Anti-mouse CD24	eBioscience	XX-0242-YY*	Clone M1/69
Anti-mouse TCRβ	eBioscience	XX-5961-YY*	Clone H57-597
Anti-mouse CD69 Biotinylated	eBioscience	13-0691-YY*	Clone H1.2F3
Anti-mouse CD5 Biotinylated	eBioscience	13-0051-YY*	Clone 53-7.3
Streptavidin	eBioscience	XX-4217-YY*
Flow cytometer	BD Biosciences - FACS Canto	338962
FACS tubes	BD Biosciences	352052
Flow cytometry analysis software	TreeStar - Flowjo	FlowJo v7/9
HyClone RPMI - 1640 medium	Thermo Scientific	SH30027.01
*XX varies by fluorochrome and YY varies by vial size.