In Situ Detectie van autoreactieve CD4 T-cellen in de hersenen en het hart met behulp van Hoofdhistocompatibiliteitscomplex Klasse II Dextramers
Summary
Is beschreven het protocol tot zelf-reactieve CD4 T-cellen te detecteren in de hersenen en het hart door directe kleuring met hoofdhistocompatibiliteitscomplex klasse II dextramers in dit rapport. Voor uitgebreide analyse is een betrouwbare methode om de frequentie van antigeen-specifieke CD4 + T-cellen in situ sommen ook ontwikkeld.
Abstract
This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-α (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler’s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the ‘Z’ serial images.
Introduction
Gebruik van major histocompatibility complex (MHC) klasse II tetrameren voor de detectie van antigeen-specifieke CD4 T-cellen is een uitdaging geweest 1-7. Om de gevoeligheid van MHC klasse II tetrameren verbeteren, heeft het onderzoeksteam gecreëerd volgende generatie tetrameren, aangeduid "dextramers" 8. Dextramers bevatten dextran ruggengraten waarbij meerdere streptavidine-fluorofoor resten geconjugeerd, zodanig dat verschillende peptide-gebonden, gebiotinyleerde MHC monomeren kan worden bevestigd aan een dextran-molecuul 9. Zo kunnen grote aggregaten van MHC dextramers dat verschillende MHC-peptidecomplexen bevatten interageren met meerdere T-celreceptoren (TCR).
Deze groep recent bekomen dextramers voor diverse zelf-antigenen en aangetoond dat de detectiegevoeligheid van dextramers ongeveer vijf keer meer dan tetrameren 8. De experimentele systemen zijn experimentele autoimmuun encefalomyelitis (EAE) geïnduceerd met proteolipid eiwit (PLP) 139-151 in SJL muizen; EAE geïnduceerd met myeline oligodendrocyt glycoproteïne (MOG) 35-55 in C57BL / 6 muizen; en experimentele auto-immune myocarditis (EAM) geïnduceerd met cardiale myosine zware keten-α (Myhc) 334-352 in A / J muizen. Deze studie demonstreert het gebruik van PLP 139-151 – en Myhc 334-352 dextramers antigeen-specifieke CD4 T-cellen te detecteren in situ met specificiteit Door een directe kleuring protocol, waardoor de noodzaak om de signalen te versterken gebruiken fluorofoor antilichamen, een benaderen gewoonlijk toegepast met behulp van tetrameren. Daarnaast is een uitgebreide methode evalueren weefsels ook bedacht betrouwbaar sommen de frequentie van antigeen-specifieke CD4 T-cellen in situ 10. Detectie van antigeen-specifieke T-cellen in situ maakt afbakening van gebeurtenissen veroorzaakt door specifieke antigene stimuli van die veroorzaakt door omstanders activatie. Ook de in situ techniek ook provides mogelijkheden om de kinetiek van antigeen-specifieke T cel infiltratie in de doelorganen volgen.
Protocol
Representative Results
Discussion
Unlike MHC klasse I tetrameren, het gebruik van MHC klasse II tetrameren voor routinetoepassingen blijft uitdagend, vooral voor de voorloper frequenties van lage affiniteit autoreactieve CD4 T-cellen, mede door hun activering afhankelijkheid 6,15-17 sommen. Onlangs werd dit probleem omzeild door het creëren van de volgende generatie van tetrameren, genaamd "dextramers," waardoor wij antigeen-specifieke CD4 T-cellen te detecteren voor een aantal auto-antigenen, zoals PLP 139-151, MOG 35-55 en Myhc 3…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dit werk werd ondersteund door de American Heart Association en de Children's Cardiomyopathie Foundation (SDG2462390204001), en de National Institutes of Health (HL114669). CM is een ontvanger van een postdoctoraal research fellowship subsidie van de Myocarditis Foundation, NJ.
Materials
| CFA | Sigma Aldrich, St Louis, MO | 5881 | Store at 4οC |
| MTB H37Rv extract | Difco Laboratories, Detroit, MI | 231141 | Store at 4οC |
| PT | List Biologicals Laboratories, Campbell, CA | 181 | Store at 4οC |
| 1x PBS | Corning, Manassas, VA | 21-040-CV | Store at 4οC |
| Female A/J mice | Jackson Laboratories, Bar Harbor, ME | 646 | |
| Female SJL/J mice | Jackson Laboratories, Bar Harbor, ME | 686 | |
| Leur-lok sterile 1 ml syrringe | BD, Franklin Lakes, NJ | 309628 | |
| Leur-lok sterile 3 ml syrringe | BD, Franklin Lakes, NJ | 309657 | |
| Sterile needle, 18 G | BD, Franklin Lakes, NJ | 305195 | |
| Sterile needle, 27 1/2 G | BD, Franklin Lakes, NJ | 305109 | |
| 3-way stopcock | Smiths Medical ASD, Inc. Dublin, OH | MX5311L | |
| Kerlix gauze bandage rolls | Covidien, Mansfield, MA | 6720 | |
| Kimwipes | Kimberly-Clark Professional, Roswell, GA | 34155 | |
| Agarose, Low Melting Point, Analytical Grade | Promega corporation, Madison, WI | V2111 | |
| Immunopure normal goat serum | Thermo Scientific, Waltham, MA | 31873 | Store at -20 οC |
| Anti-mouse CD4 conjugated with PE dye | eBioscience, San Diego, CA | 12004185 | Store at 4οC |
| Paraformaldehyde | Electron Microscopy Sciences, Hatfield, PA | 19202 | Store at 4οC |
| Faramount Aqueous Mounting Medium | Dako, Carpinteria, CA | 10073021 | |
| Conical tube (15ml) | BD Biosciences, San Jose, CA | 352099 | |
| Conical tube (50 ml) | BD Biosciences, San Jose, CA | 352070 | |
| 24-well flat bottomed tissue culture plates | BD Biosciences, San Jose, CA | 356775 | |
| Water color #2 brushes | Charles Leonard, Inc. Hauppauge, NY | 73502 | |
| Plain Microscope slides, size: 3" x 1" x 1.2 mm | Fisher Scientific, Pittsburgh, PA | 1255010 | |
| Premium Cover glass, 22 x 22-1 | Fisher Scientific, Pittsburgh, PA | 12548B | |
| Disposable deep base mold histology cassettes | Laboratory prodcust, Rochester, NY | M-475-10 | |
| Loctite Super glue | Henkel Corporation, Westlake, OH | 1471879 | |
| Razor blades | Gillette SuperSilver | ||
| Myhc-a 334-352 (DSAFDVLSFTAEEKAGVYK) | Neopeptide, Cambridge, MA | Store at 4οC | |
| PLP 139-151 (HSLGKWLGHPDKF) | Neopeptide, Cambridge, MA | Store at 4οC | |
| MHC class II/IAs PLP 139-151 or TMEV 70-86 dextramers conjugated with APC dye | Store at 4οC | ||
| MHC class II/IAk Myhc 334-352 or or RNase 40-56 dextramers conjugated with APC dye | Store at 4οC | ||
| Dextran conjugated SA-APC | Immundex, Copenhagen, Demark | Store at 4οC | |
| Sterile surgical scissors and forceps | INOX tool Corporation | ||
| Micro oven | GE Healthcare, Pittsuburgh, PA | ||
| Leica VT 1200 Vibratome | Leica Microsystems, Inc. Buffalo Grove, IL | ||
| Olympus BX 60 Laser scanning confocal microscope | Olympus America, Inc. Center Valley, PA | ||
| Nikon A1-Eclipse 90i confocal microscope | Nikon Inc. Melville, NY |
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