Summary

Whole-dyr Imaging og Flowcytometrisk Teknikker til analyse af antigen-specifikke CD8 + T-celle responser efter Nanopartikel Vaccination

Published: April 29, 2015
doi:

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

Alle forsøg er beskrevet i denne protokol, blev godkendt af University Udvalg for Anvendelse og pleje af dyr (UCUCA) ved University of Michigan og udført i overensstemmelse med de fastlagte politikker og retningslinjer. 1. Syntese og karakterisering af ICMVs Co-loaded med proteinantigen og adjuvansmolekyler Bland 1: 1 molært forhold af 1,2-dioleoyl–sn-glycero-3-phosphocholin (DOPC) og 1,2-dioleoyl–sn-glycero-3-phosphoethanolamine- N – [4- (p -maleim…

Representative Results

De involverede i syntesen af ICMVs trin er illustreret i figur 1 6. Kort fortalt en lipidfilm indeholdende eventuelle lipofile lægemidler eller fluorescerende farvestoffer hydreret i nærværelse af hydrofile lægemidler. Divalente kationer, såsom Ca2 +, tilsættes til at drive fusion af anioniske liposomer i multilamellære vesikler. Dithiol tværbinder, såsom DTT, sættes til "fibre" maleimid-funktionaliserede lipider på apposing lipidlag, og endelig resterende ekst…

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

References

  1. Irvine, D. J., Swartz, M. A., Szeto, G. L. Engineering synthetic vaccines using cues from natural immunity. Nature materials. 12, 978-990 (2013).
  2. Moon, J. J., Huang, B., Irvine, D. J. Engineering nano- and microparticles to tune immunity. Advanced materials. 24, 3724-3746 (2012).
  3. Sahdev, P., Ochyl, L. J., Moon, J. J. Biomaterials for nanoparticle vaccine delivery systems. Pharmaceutical Research. , (2014).
  4. Zhao, L., et al. Nanoparticle vaccines. Vaccine. 32, 327-337 (2014).
  5. Riet, E., Ainai, A., Suzuki, T., Kersten, G., Hasegawa, H. Combatting infectious diseases; nanotechnology as a platform for rational vaccine design. Advanced drug delivery reviews. 74C, 28-34 (2014).
  6. Moon, J. J., et al. Interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses. Nature Materials. 10, 243-251 (2011).
  7. Moon, J. J., et al. Enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand Tfh cells and promote germinal center induction. Proceedings of the National Academy of Sciences of the United States of America. 109, 1080-1085 (2012).
  8. DeMuth, P. C., Moon, J. J., Suh, H., Hammond, P. T., Irvine, D. J. Releasable layer-by-layer assembly of stabilized lipid nanocapsules on microneedles for enhanced transcutaneous vaccine delivery. ACS Nano. 6, 8041-8051 (2012).
  9. Li, A. V., et al. Generation of Effector Memory T Cell-Based Mucosal and Systemic Immunity with Pulmonary Nanoparticle Vaccination. Science Translational Medicine. 5, 204ra130 (2013).
  10. Stephan, M. T., Moon, J. J., Um, S. H., Bershteyn, A., Irvine, D. J. Therapeutic cell engineering with surface-conjugated synthetic nanoparticles. Nature Medicine. 16, 1035-1041 (2010).
  11. Murdock, R. C., Braydich-Stolle, L., Schrand, A. M., Schlager, J. J., Hussain, S. M. Characterization of nanomaterial dispersion in solution prior to In vitro exposure using dynamic light scattering technique. Toxicological Sciences. 101, 239-253 (2008).
  12. Manolova, V., et al. Nanoparticles target distinct dendritic cell populations according to their size. European Journal of Immunology. 38, 1404-1413 (2008).
  13. Reddy, S. T., et al. Exploiting lymphatic transport and complement activation in nanoparticle vaccines. Nature Biotechnology. 25, 1159-1164 (2007).
  14. Kaur, R., Bramwell, V. W., Kirby, D. J., Perrie, Y. Manipulation of the surface pegylation in combination with reduced vesicle size of cationic liposomal adjuvants modifies their clearance kinetics from the injection site, and the rate and type of T cell response. Journal of Controlled Release. 164, 331-337 (2012).
  15. Zhuang, Y., et al. PEGylated cationic liposomes robustly augment vaccine-induced immune responses: Role of lymphatic trafficking and biodistribution. Journal of Controlled Release. 159, 135-142 (2012).
  16. Hogquist, K. A., et al. T cell receptor antagonist peptides induce positive selection. Cell. 76, 17-27 (1994).
  17. Clarke, S. R. M., et al. Characterization of the ovalbumin-specific TCR transgenic line OT-I: MHC elements for positive and negative selection. Immunology and Cell Biology. 78, 110-117 (2000).
  18. Azadniv, M., Dugger, K., Bowers, W. J., Weaver, C., Crispe, I. N. Imaging CD8(+) T cell dynamics in vivo using a transgenic luciferase reporter. International Immunology. 19, 1165-1173 (2007).
  19. Kim, D., Hung, C. F., Wu, T. C. Monitoring the trafficking of adoptively transferred antigen- specific CD8-positive T cells in vivo, using noninvasive luminescence imaging. Human gene therapy. 18, 575-588 (2007).
  20. Rabinovich, B. A., et al. Visualizing fewer than 10 mouse T cells with an enhanced firefly luciferase in immunocompetent mouse models of cancer. Proceedings of the National Academy of Sciences of the United States of America. 105, 14342-14346 (2008).
  21. Altman, J. D., et al. Phenotypic analysis of antigen-specific T lymphocytes. Science. 274, 94-96 (1996).
  22. Machholz, E., Mulder, G., Ruiz, C., Corning, B. F., Pritchett-Corning, K. R. Manual Restraint and Common Compound Administration Routes in Mice and Rats. Journal of Visualized Experiments. , e2771 (2012).
  23. Bedoya, S. K., Wilson, T. D., Collins, E. L., Lau, K., Larkin Iii, J. Isolation and Th17 Differentiation of Naive CD4 T Lymphocytes. Journal of Visualized Experiments. , e50765 (2013).
  24. Chen, Y., et al. Visualization of the interstitial cells of cajal (ICC) network in mice. Journal of Visualized Experiments. , (2011).
  25. Scheffold, A., Busch, D. H., Kern, F., Sack, U., Tárnok, D. H., Rothe, G. In Cellular Diagnostics Basics, Methods and Clinical Applications of Flow Cytometry. Karger. , 476-502 (2009).
  26. Wilson, K., Yu, J., Lee, A., Wu, J. C. In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells. Journal of Visualized Experiments. , e740 (2008).
  27. Golde, W. T., Gollobin, P., Rodriguez, L. L. A rapid, simple, and humane method for submandibular bleeding of mice using a lancet. Lab Animal. 34, 39-43 (2005).
  28. Tilney, N. L. Patterns of lymphatic drainage in the adult laboratory rat. Journal of Anatomy. 109, 369-383 (1971).
  29. Stivaktakis, N., et al. PLA and PLGA microspheres of beta-galactosidase: Effect of formulation factors on protein antigenicity and immunogenicity. Journal of Biomedical Materials Research Part A. 70A, 139-148 (2004).
  30. Bilati, U., Allemann, E., Doelker, E. Nanoprecipitation versus emulsion-based techniques for the encapsulation of proteins into biodegradable nanoparticles and process-related stability issues. AAPS PharmSciTech. 6, E594-E604 (2005).
  31. Blander, J. M., Medzhitov, R. Toll-dependent selection of microbial antigens for presentation by dendritic cells. Nature. 440, 808-812 (2006).
  32. Iwasaki, A., Medzhitov, R. Regulation of adaptive immunity by the innate immune system. Science. 327, 291-295 (2010).
  33. Dubochet, J., et al. Cryo-electron microscopy of vitrified specimens. Quarterly reviews of biophysics. 21, 129-228 (1988).
  34. Vinson, P. K., Talmon, Y., Walter, A. Vesicle-micelle transition of phosphatidylcholine and octyl glucoside elucidated by cryo-transmission electron microscopy. Biophysical Journal. 56, 669-681 (1989).
  35. Schagger, H. Tricine-SDS-PAGE. Nature Protocols. 1, 16-22 (2006).
  36. Chevallet, M., Luche, S., Rabilloud, T. Silver staining of proteins in polyacrylamide gels. Nat Protoc. 1, 1852-1858 (2006).
  37. Randolph, G. J., Angeli, V., Swartz, M. A. Dendritic-cell trafficking to lymph nodes through lymphatic vessels. Nature Reviews Immunology. 5, 617-628 (2005).
  38. Liu, H., et al. Structure-based programming of lymph-node targeting in molecular vaccines. Nature. 507, 519-522 (2014).
  39. Xu, Z., et al. Multifunctional nanoparticles co-delivering Trp2 peptide and CpG adjuvant induce potent cytotoxic T-lymphocyte response against melanoma and its lung metastasis. Journal of Controlled Release. 172, 259-265 (2013).
  40. Hailemichael, Y., et al. Persistent antigen at vaccination sites induces tumor-specific CD8(+) T cell sequestration, dysfunction and deletion. Nature Medicine. 19, 465 (2013).
  41. Slingluff, C. L., et al. Phase I trial of a melanoma vaccine with gp100(280-288) peptide and tetanus helper peptide in adjuvant: Immunologic and clinical outcomes. Clinical Cancer Research. 7, 3012-3024 (2001).
  42. Speiser, D. E., et al. Rapid and strong human CD8(+) T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909. Journal of Clinical Investigation. 115, 739-746 (2005).
  43. Araki, K., et al. mTOR regulates memory CD8 T-cell differentiation. Nature. 460, 108-112 (2009).
  44. Masopust, D., Vezys, V., Marzo, A. L., Lefrancois, L. Preferential localization of effector memory cells in nonlymphoid tissue. Science. 291, 2413-2417 (2001).
  45. Cuburu, N., et al. Intravaginal immunization with HPV vectors induces tissue-resident CD8+ T cell responses. Journal of Clinical Investigation. 122, 4606-4620 (2012).
  46. Ahlers, J. D., Belyakov, I. M. Memories that last forever: strategies for optimizing vaccine T-cell memory. Blood. 115, 1678-1689 (2010).
  47. Seder, R. A., Darrah, P. A., Roederer, M. T-cell quality in memory and protection: implications for vaccine design. Nature Reviews Immunology. 8, 247-258 (2008).
  48. Barber, D. L., et al. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature. 439, 682-687 (2006).
  49. Wherry, E. J., et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity. 27, 670-684 (2007).
  50. Czerkinsky, C. C., Nilsson, L. A., Nygren, H., Ouchterlony, O., Tarkowski, A. A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. Journal of Immunological Methods. 65, 109-121 (1983).
  51. Foster, B., Prussin, C., Liu, F., Whitmire, J. K., Whitton, J. L. Detection of intracellular cytokines by flow cytometry. Current Protocols in Immunology. Chapter 6 (Unit 6 24), (2007).
  52. Wonderlich, J., Shearer, G., Livingstone, A., Brooks, A. Induction and measurement of cytotoxic T lymphocyte activity. Current protocols in immunology. Chapter 3 (Unit 6 24), (2006).
  53. Betts, M. R., et al. Sensitive and viable identification of antigen-specific CD8+T cells by a flow cytometric assay for degranulation. Journal of Immunological Methods. 281, 65-78 (2003).
  54. Brunner, K. T., Mauel, J., Cerottini, J. C., Chapuis, B. Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology. 14, 181-196 (1968).
  55. Noto, A., Ngauv, P., Trautmann, L. Cell-based flow cytometry assay to measure cytotoxic activity. Journal of Visualized Experiments. , e51105 (2013).
  56. Quah, B. J., Wijesundara, D. K., Ranasinghe, C., Parish, C. R. The use of fluorescent target arrays for assessment of T cell responses in vivo. Journal of visualized experiments. , e51627 (2014).
  57. Crotty, S. Follicular helper CD4 T cells (TFH). Annual review of immunology. 29, 621-663 (2011).

Play Video

Cite This Article
Ochyl, L. J., Moon, J. J. Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination. J. Vis. Exp. (98), e52771, doi:10.3791/52771 (2015).

View Video