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 genita....

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

List of materials used in this article

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.