Whole-dyr Imaging og Flowcytometrisk Teknikker til analyse af antigen-specifikke CD8 + T-celle responser efter Nanopartikel Vaccination
Summary
We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.
Abstract
Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a “pathogen-mimicking” nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.
Introduction
Traditionel vaccine udvikling har primært ansat den empiriske tilgang trial-and-error. , Med den seneste udvikling i en bred vifte af biomaterialer og opdagelsen af molekylære determinanter for immun aktivering, er det nu muligt at rationelt designe vaccineformuleringer med biofysiske og biokemiske signaler afledt af patogener 1,2. Især har forskellige partikelformige drug delivery platforme blevet undersøgt som vaccine-bærere, som de kan co-loaded med subunit-antigener og immunstimulerende midler, beskytte vaccinekomponenter mod nedbrydning, og øge deres co-levering til antigenpræsenterende celler (APC'er) bopæl i lymfeknuder knuder (LNS), dermed maksimere immun stimulering og aktivering 3-5. I denne rapport beskriver vi syntesen af en "patogen-efterligne" nanopartikel-system, betegnes interbilayer-tværbundet multilamellare vesikler (ICMVs), som tidligere er blevet påvist som en potent vaccine platform;m for fremkaldelse af robust cytotoksisk T-lymfocyt (CTL) og humorale immunresponser i både systemiske og mucosale vævsrum 6-9. Især opnåede vaccination med ICMVs væsentligt forbedret serum IgG-niveauer mod en malaria-antigen, sammenlignet med vaccination med konventionelle adjuvanser (fx alun og Montanide) 7 og også fremkaldte kraftige CTL-responser mod tumorceller og viral challenge-modeller i mus 9. Her, ved hjælp ICMVs som model vaccine nanopartikel-system beskriver vi metoder til karakterisering af vaccine nano-formuleringer, herunder partikelstørrelse og zeta potentielle målinger og sporing af partikel menneskehandel til drænende LN (DLN'er) udnytte konfokal billeddannelse af cryosectioned væv 7. Derudover præsenterer vi en hel-dyr imaging-baseret fremgangsmåde til analyse af udvidelse af CTL-responser i mus efter adoptiv overførsel af luciferase-udtrykkende antigen-specifikke CD8 + T-celler 9,10. Endelig har vi deSkriveren tetramerfarvning af perifere mononukleære blodceller (PBMC'er) for langsgående kvantificering af endogene T-celle-reaktioner i mus vaccineret med nanopartikler 6,9.
ICMVs er et lipid-baserede nanopartikel formulering syntetiseret ved kontrolleret fusion af simple liposomer i multilamellære strukturer, som derefter kemisk stabiliseret ved tværbinding maleimid-funktionaliseret phospholipid hovedgrupper inden lipidlag med dithiol tværbindere 6. Når ICMVs syntetiseres, kan en lille brøkdel af nanopartikler anvendes til at bestemme partikelstørrelse og zeta-potentialet (dvs. overfladeladningen af partikler) med en (DLS) systemet dynamisk lysspredning og et zeta-potentialet analysator. DLS måler ændringer i lysspredning i partikel suspensioner, der tillader bestemmelse af diffusionskoefficienten og den hydrodynamiske størrelse af partikler 11. Opnåelse konsekvent partikelstørrelse fra parti til parti-syntese er kritiskeftersom partikelstørrelsen er en af de vigtigste faktorer, der påvirker lymfe dræning af vaccine partikler DLN'er og efterfølgende cellulær optagelse af APC'er 12,13. Derudover kan zetapotentialet opnås ved måling partikelhastigheden når der påføres en elektrisk strøm, hvilket muliggør bestemmelse af den elektroforetiske mobilitet af partikler og partikel overfladeladning 11. Er vigtigt at sikre konsistente Zetapotential værdier for partikler, da overfladeladning af partikler bestemmer kolloid stabilitet, som har direkte indvirkning på partikeldispersion under opbevaring og efter in vivo administration 14,15. For at spore partiklen lokalisering til DLN'er, kan ICMVs mærkes med ønskede fluoroforer herunder lipofile farvestoffer og kovalent mærkede antigener. Efter immunisering kan mus aflives på forskellige tidspunkter, DLN'er resekteret, cryosectioned og analyseret med konfokal mikroskopi. Denne teknik muliggør visualisering af lymfatisk Dralning af begge nanopartikel vaccine luftfartsselskaber og antigen til DLN'er. Vævssnittene kan yderligere farvet med fluorescensmærkede antistoffer og anvendes til at opnå mere information, såsom typer af celler associeret med antigenet og dannelse af kimcentre som vi har vist tidligere 7.
Når partiklen syntese er optimeret og handel til DLN'er bekræftes, er det vigtigt at validere fremkaldelse af in vivo CTL ekspansion. For at analysere fremkaldelse af antigenspecifikke CD8 + -T-celler som respons på vaccination nanopartikel, har vi udnyttet en model antigen, ovalbumin (OVA), med OVA 257-264 peptid (SIINFEKL) immundominerende CD8 + T-celleepitop, som giver detaljerede immunologiske analyser af antigenspecifikke T celleresponser for den første vaccineudvikling 16,17. Navnlig at udspørge dynamikken i ekspansion og migration af antigenspecifikke CD8 + -T-celler, har vi skabt endobbelt-transgen musemodel ved at krydse ildflue luciferase-udtrykkende transgene mus (Luc) med OT-I transgene mus, som besidder CD8 + T-celler med T-celle receptor (TCR) specifik for SIINFEKL (i association med H-2K b). Fra disse OT-I / Luc-mus, luciferase-udtrykkende, OT-I CD8 + -T-celler kan isoleres og forberedes til adoptiv overførsel til naive C57BL / 6-mus. Når seeded vil vellykket immunisering med OVA-indeholdende nanopartikler resultere i udvidelse af de overførte T-celler, som kan spores ved overvågning af bioluminescens signal med et helt dyr imaging system 9,10. Denne ikke-invasive hele kroppen imaging teknik har været anvendt med andre virale eller tumorantigener i fortiden 18-20, afslører processer involveret i T-celle-ekspansion i lymfevæv og formidling til perifere væv i en langsgående måde.
Komplementær til analyse af adoptivt overført antigenspecifikke CD8 + -T-celler, endogenoos T-cellereaktioner efter vaccination kan undersøges med peptidet-major histocompatibility complex (MHC) tetramer assay 21, hvori en peptid-MHC tetramer-kompleks, bestående af fire fluorofor-mærkede MHC klasse I molekyler fyldt med peptid-epitoper, anvendes at binde TCR og label CD8 + T-celler i en antigen-specifik måde. Peptid-MHC tetramer-assay kan udføres enten i terminale nekropsi undersøgelser for at identificere antigenspecifikke CD8 + T-celler i lymfoide og perifere væv eller i langsgående undersøgelser med perifere mononukleære blodceller (PBMC'er) opnået fra seriel blod trækker. Efter farvning lymfocytter med peptid-MHC tetramer, flowcytometri analyse udføres for detaljerede analyser af fænotypen af CTL'er eller kvantificering af deres hyppighed blandt CD8 + T-celler.
Protocol
Representative Results
Discussion
Oplysningerne i denne artikel protokol beskriver syntese og karakterisering af en ny lipidbaseret nanopartikel-system, kaldet ICMVs, og giver processen med validering effektiviteten af nanopartikler-baserede vaccineformuleringer at fremkalde antigen-specifikke CD8 + T-celle responser. ICMV syntese er afsluttet i alle vandige tilstand, hvilket er en stor fordel i forhold til andre almindeligt anvendte polymere nanopartikler systemer (fx poly (lactid-co-glycolid) sure partikler), der typisk kræver organisk…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Denne undersøgelse blev støttet af National Institute of Health giver 1K22AI097291-01 og af National Center for Fremme Translationelle Sciences i National Institutes of Health i henhold Award nummer UL1TR000433. Vi anerkender også Prof. Darrell Irvine på MIT og Prof. Matthias Stephan på Fred Hutchinson Cancer Center for deres bidrag på det indledende arbejde på vaccine nanopartikler og OT-I / Luc transgene mus.
Materials
| 1. Synthesis and characterization of ICMVs co-loaded with protein antigen and adjuvant molecules | |||
| 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (sodium salt) (MPB) | Avanti Polar Lipids, INC. | 870012 | |
| 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) | Avanti Polar Lipids, INC. | 850375 | |
| Monophosphoryl Lipid A (Synthetic) (PHAD™) (MPLA) | Avanti Polar Lipids, INC. | 699800 | |
| 20 mL glass vials | Wheaton | 0334125D | |
| Symphny Vacuum Oven | VWR | 414004-580 | |
| Ovalbumin (OVA) | Worthington | 3054 | |
| Bis-Tris Propane (BTP) | Fisher | BP2943 | |
| Q125 Sonicator (125W/20kHz) | Qsonica | Q125-110 | |
| Dithiothreitol (DTT) | Fisher | BP172 | |
| 2 kDa Thiolated Polyethylene Glycol (PEG-SH) | Laysan Bio | MPEG-SH-2000-1g | |
| Malvern ZetaSizer Nano ZSP | Malvern | ||
| ZetaSizer Cuvettes | Malvern | DTS1070 | |
| 2. Examination of lymph node draining of fluorescence-tagged ICMVs with confocal microscopy | |||
| 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt (DID) | Life Technologies | D-7757 | |
| Alexa Fluor 555-succinimidyl ester (AF555-NHS) | Life Technologies | A37571 | |
| Tissue-Tek OCT freezing medium | VWR | 25608-930 | |
| Tissue Cryomolds | VWR | 25608-922 | |
| 3. Monitoring expansion of antigen-specific, luciferase-expressing CD8+ T cells after nanoparticle vaccination with whole animal imaging | |||
| C57BL/6 mice | Jackson | 000664 | |
| Albino C57BL/6 mice | Jackson | 000058 | |
| OT-1 C57BL/6 mice | Jackson | 003831 | |
| 70 μm nylon strainer | BD | 352350 | |
| EasySep™ Mouse CD8+ T Cell Isolation Kit | StemCell | 19853 | |
| IVIS® whole animal imaging system | Perkin Elmer | ||
| 4. Peptide-MHC tetramer staining of peripheral blood mononuclear cells (PBMCs) for flow cytometric analysis of antigen-specific CD8+ T cells | |||
| K2EDTA tubes | BD | 365974 | |
| ACK lysis buffer | Life Technologies | A10492-01 | |
| Anti-CD16/32 Fc Block | Ebioscience | 14-0161-86 | |
| H-2Kb OVA Tetramer | MBL | TS-5001-1C | |
| Anti-CD8-APC | BD | 553031 | |
| Anti-CD44-FITC | BD | 553133 | |
| Anti-CD62L-PECy7 | Ebioscience | 25-0621-82 | |
| 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) | SIGMA | D8417-10MG | |
| CyAn Flow Cytometer | Beckman Coulter | ||
| FlowJo Software | FlowJo |
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