Undersøgelse af tymisk positiv og negativ selektion ved flowcytometri
Summary
Vi præsenterer en flowcytometri-baserede metode til at undersøge T-celleudvikling<em> In vivo</em> Hjælp af genetisk manipulerede mus på en vildtype-eller T-cellereceptor transgen baggrund.
Abstract
Et sundt immunsystem kræver, at T-celler reagerer på fremmede antigener, mens de resterende tolerant til selv-antigener. Tilfældig omlejring af T-cellereceptoren (TCR) α og β loci genererer et T-celle-repertoire med stor forskel på antigenspecificitet, både til sig selv og fremmede. Udvælgelse af repertoiret under udvikling i thymus er kritisk for generering af sikre og egnede T-celler. Defekter i thymisk udvælgelse bidrage til udviklingen af autoimmune og immundefekt lidelser 1-4.
T-celle-progenitorer ind thymus som dobbelt-negative (DN) thymocytter, som ikke udtrykker CD4-eller CD8-co-receptorer. Ekspression af αβTCR og begge coreceptorer forekommer ved den dobbelte positive (DP) fase. Interaktion af αβTCR med selv-peptid-MHC (pMHC) præsenteret ved thymiske celler bestemmer skæbnen for DP thymocyt. Høj affinitet interaktioner fører til negativ selektion og elimineringtion af selvnedbrydende thymocytter. Lav affinitet interaktioner resulterer i positiv selektion og udvikling af CD4 eller CD8 enkelte positive (SP) T-celler stand til at genkende fremmede antigener præsenteret af selv-MHC 5.
Positiv selektion kan undersøges i mus med et polyklonalt (vildtype) TCR repertoire ved at observere dannelsen af modne T-celler. De er imidlertid ikke ideelle til studiet af negativ udvælgelse, der involverer deletion af små antigen-specifikke populationer. Mange modelsystemer har været anvendt til at undersøge negativ selektion, men varierer i deres evne til at rekapitulere fysiologiske begivenheder 6. For eksempel mangler in vitro-stimulering af thymocytter den thymiske miljø, som er tæt involveret i selektion, mens indgivelse af exogent antigen kan føre til ikke-specifik deletion af thymocytter 7-9. I øjeblikket er de bedste værktøjer til at studere in vivo negativ selektion er mus, der udtrykker en transgENIC TCR specifikke for endogene selv-antigen. Imidlertid er mange klassiske TCR-transgene modeller karakteriseret ved for tidlig ekspression af det transgene TCRα kæde på DN fase, hvilket resulterer i for tidlig negativ selektion. Vores laboratorium har udviklet HY CD4 model, hvor det transgene HY TCRα er betinget udtrykt på DP fase, hvilket tillader negativ selektion til at forekomme i DP til SP overgang som forekommer i vildtype-mus 10.
Her beskriver vi en flowcytometri-baseret protokol til at undersøge thymisk positiv og negativ selektion i HY CD4 musemodel. Mens negativ selektion i HY CD4 mus er meget fysiologisk, kan disse fremgangsmåder også anvendes på andre TCR-transgene modeller. Vi vil også præsentere generelle strategier til at analysere positiv selektion i et polyklonalt repertoire gælder for genetisk manipulerede mus.
Protocol
Representative Results
Discussion
Protokollen præsenteres her kan anvendes til at undersøge positiv og negativ selektion i ikke-TCR transgene og TCR-transgene mus. Denne protokol beskrives farvning af overflade-antigener. For en nærmere analyse af molekylære mekanismer, er det ofte nødvendigt at udføre intracellulær farvning. Vi bruger BD Biosciences Cytofix / Cytoperm Kit for de fleste intracellulære proteiner og BD Biosciences foxp3 farvningssæt for transkriptionsfaktorer. Vi plejer at købe vores prøver umiddelbart efter farvning. Imidlerti…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gerne takke Bing Zhang for hans teknisk bistand. Dette arbejde blev finansieret af den canadiske Institutes for Health Research (MOP-86.595). TAB er en CIHR New Investigator og AHFMR Scholar. QH er understøttet af en CIHR Canada Graduate Scholarship – Ph.d. og en AIHS Fuldtids studentship. SAN er understøttet af en dronning Elizabeth II Graduate Scholarship. AYWS understøttes af en NSERC Postgraduate Scholarship – Doctoral.
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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