Summary

Экспертиза тимуса позитивной и негативной селекции с помощью проточной цитометрии

Published: October 08, 2012
doi:

Summary

Мы представляем проточной цитометрии на основе метода для изучения Т-клеточного развития<em> В естественных условиях</em> С использованием генетически модифицированных мышей дикого типа или Т-клеточного рецептора трансгенных фоне.

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 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 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 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 TCRα chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 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 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.

Protocol

См. рисунок 1 для общей схемы экспериментальных протоколов. 1. Диссекция Место стерильной сито стали в 60 х 15 мм блюдо Петри. Одна единица необходима в образце ткани. Добавить 5 мл сбалансированного солевого раствора Хэнкса (HBSS) к каждому блюду. Держите б…

Representative Results

В физиологических TCR модели трансгенных мышей дикого типа и, положительный отбор начинается с DP яркий этап перед переходом в DP скучным этапом после встречи антигена. DP скучно тимоцитов затем ввести переходный CD4 + CD8 вот этапе, прежде чем стать CD4SP или CD8SP тим?…

Discussion

Протокол, представленные здесь может быть использован для изучения положительного и отрицательного отбора в не-TCR трансгенных и TCR трансгенных мышей. Этот протокол описывает окрашивания поверхностных антигенов. Для дальнейшего анализа молекулярных механизмов, часто бывает необходим…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Авторы хотели бы поблагодарить Bing Zhang за техническую помощь. Эта работа финансировалась Канадский институт исследований в области здравоохранения (MOP-86595). TAB является следователь CIHR новые и AHFMR Scholar. QH поддерживается CIHR Канаде Высшее Стипендия – докторских и AIHS Полный рабочий день студенчества. SAN при поддержке королевы Елизаветы II Высшее стипендии. AYWS поддерживается NSERC последипломного Стипендия – докторантуры.

Materials

Name of the reagent Company Catalogue number Comments (optional)
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.

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Cite This Article
Hu, Q., Nicol, S. A., Suen, A. Y., Baldwin, T. A. Examination of Thymic Positive and Negative Selection by Flow Cytometry. J. Vis. Exp. (68), e4269, doi:10.3791/4269 (2012).

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