Name: rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development
Uploaded: 2018-06-19T13:00:00
Description: Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

Vi står i gjeld til våre tekniske assistenter: U. Bröder og H. Shkarlet, som hjalp oss under eksperimentelle prosedyrer. Dette arbeidet var delvis finansiert av EU tilskudd (UniVax, kontrakt nr. 601738 og TRANSVAC2, kontrakt nr. 730964) og en Helmholtz Association grant (HAI-IDR). Finansieringskilder ikke påvirke forskning design, generasjon av manuskriptet eller beslutning om å sende det til publikasjonen.

Alle mus brukt i denne studien var fra C57BL/6 bakgrunnen. Alle dyrene ble holdt under patogen-gratis forhold. Alle eksperimentene ble utført etter den normative tyske Dyrevern loven (TierSchG BGBl. JEG S 1105; 25.05.1998) og ble godkjent av lavere Sachsen komité for etikk av dyr eksperimenter og staten kontoret (lavere Niedersachsen State Office of Consumer Protection og Food Safety), arbeidstillatelse nummer 33,4-42502-04-13/1281 og 162280.
1. CFSE farging av OT-jeg T celler og adopsjon over...

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+delivery+of+a+respiratory+syncytial+virus+CTL+peptide+with+enterotoxin-based+adjuvants+elicits+protective,+immunopathogenic,+and+immunoregulatory+antiviral+CD8++T+cell+responses.">Mucosal delivery of a respiratory syncytial virus CTL peptide with enterotoxin-based adjuvants elicits protective, immunopathogenic, and immunoregulatory antiviral CD8+ T cell responses.</a> Journal of immunology. 166, 1106-1113 (2001).</li><li>Fulginiti, V. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+Virus+Immunizationa+Field+Trial+Of+Two+Inactivated+Respiratory+Virus+Vaccines;+An+Aqueous+Trivalent+Paratnfluenza+Virus+Vaccine+And+An+Alum-Precipitated+Respiratory+Syncytial+Virus+Vaccine1.">Respiratory Virus Immunizationa Field Trial Of Two Inactivated Respiratory Virus Vaccines; An Aqueous Trivalent Paratnfluenza Virus Vaccine And An Alum-Precipitated Respiratory Syncytial Virus Vaccine1.</a> American journal of epidemiology. 89, 435-448 (1969).</li><li>Olson, M. R., Varga, S. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+antagonist+peptides+induce+positive+selection.">T cell receptor antagonist peptides induce positive selection.</a> Cell. 76, 17-27 (1994).</li><li>Clarke, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+ovalbumin-specific+TCR+transgenic+line+OT-I:+MHC+elements+for+positive+and+negative+selection.">Characterization of the ovalbumin-specific TCR transgenic line OT-I: MHC elements for positive and negative selection.</a> Immunology and cell biology. 78, 110-117 (2000).</li><li>Topham, D. J., Castrucci, M. R., Wingo, F. S., Belz, G. T., Doherty, P. 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Part B, Clinical cytometry. 72, 8-13 (2007).</li><li>Newrzela, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+receptor+diversity+prevents+T-cell+lymphoma+development.">T-cell receptor diversity prevents T-cell lymphoma development.</a> Leukemia. 26, 2499-2507 (2012).</li><li>Iwasaki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergen+endotoxins+induce+T-cell-dependent+and+non-IgE-mediated+nasal+hypersensitivity+in+mice.">Allergen endotoxins induce T-cell-dependent and non-IgE-mediated nasal hypersensitivity in mice.</a> J Allergy Clin Immunol. 139, 258-268 (2017).</li><li>Tsuchiya, K., Siddiqui, S., Risse, P. A., Hirota, N., Martin, J. 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American journal of physiology.</a> Lung cellular and molecular physiology. , L54-L63 (2012).</li><li>Burgdorf, S., Scholz, C., Kautz, A., Tampe, R., Kurts, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+mechanistic+separation+of+cross-presentation+and+endogenous+antigen+presentation.">Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation.</a> Nature. 9, 558-566 (2008).</li><li>Last'ovicka, J., Budinsky, V., Spisek, R., Bartunkova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+lymphocyte+proliferation:+CFSE+kills+dividing+cells+and+modulates+expression+of+activation+markers.">Assessment of lymphocyte proliferation: CFSE kills dividing cells and modulates expression of activation markers.</a> Cellular immunology. , 79-85 (2009).</li><li>Oelke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+CD8(+)+antigen-specific+cytotoxic+T+lymphocytes+after+enrichment+based+on+cytokine+secretion:+comparison+with+the+MHC-tetramer+technology.">Functional characterization of CD8(+) antigen-specific cytotoxic T lymphocytes after enrichment based on cytokine secretion: comparison with the MHC-tetramer technology.</a> Scand J Immunol. 52, 544-549 (2000).</li><li>Wang, W., Golding, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytotoxic+T+lymphocyte+response+against+a+protein+antigen+does+not+decrease+the+antibody+response+to+that+antigen+although+antigen-pulsed+B+cells+can+be+targets.">The cytotoxic T lymphocyte response against a protein antigen does not decrease the antibody response to that antigen although antigen-pulsed B cells can be targets.</a> Immunology letters. 100, 195-201 (2005).</li><li>O'Sullivan, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+CD8(+)+T+cells+use+cell-intrinsic+lipolysis+to+support+the+metabolic+programming+necessary+for+development.">Memory CD8(+) T cells use cell-intrinsic lipolysis to support the metabolic programming necessary for development.</a> Immunity. 41, 75-88 (2014).</li><li>Xu, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+interferon+protects+antiviral+CD8++T+cells+from+NK+cell+cytotoxicity.">Type I interferon protects antiviral CD8+ T cells from NK cell cytotoxicity.</a> Immunity. 40, 949-960 (2014).</li><li>Volk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+three+humanized+mouse+models+for+adoptive+T+cell+transfer.">Comparison of three humanized mouse models for adoptive T cell transfer.</a> The journal of gene medicine. 14, 540-548 (2012).</li><li>Safinia, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+Mice+as+Preclinical+Models+in+Transplantation.">Humanized Mice as Preclinical Models in Transplantation.</a> Methods Mol Biol. 1371, 177-196 (2016).</li><li>Grover, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+NOG+mice+as+a+model+for+tuberculosis+vaccine-induced+immunity:+a+comparative+analysis+with+the+mouse+and+guinea+pig+models+of+tuberculosis.">Humanized NOG mice as a model for tuberculosis vaccine-induced immunity: a comparative analysis with the mouse and guinea pig models of tuberculosis.</a> Immunology. 152, 150-162 (2017).</li></ol>

Moderne vaksiner er ideelt composedof renset antigen og adjuvans, med mulig tillegg av en levering system som liposomer, virus-lignende partikler, nanopartikler eller live vektorer. Et viktig aspekt når du utformer en vaksine er å velge riktig adjuvant etter kliniske behov. Del av omfanget kan omfatte favoriserer en humoral mot mobilnettet immunforsvaret (eller begge), valget av en lokal vs en systemisk immunrespons (eller begge) og typen minne som vaksinen må generere i målgruppen. Et viktig aspekt av adjuvant evalu...

Cytotoksiske T-lymfocytter (CTL) inducing vaksiner er viktige terapeutiske tiltak som er utviklet for å bekjempe visse typer kreft1. CTL er også viktig for forebyggende vaksiner mot intracellulær patogener2. Videre CTLEN er en av de få immun forsvarsmekanismer funksjonelt aktive risiko bestander som nyfødte3,4 som også avhenger CTL å bekjempe tidlig liv infeksjoner5. I denne forbindelse resulterte vaksiner mot luftveiene Syncytial Virus (RSV) som ble utviklet med en adjuvans som ikke framprovosere CTL svar (Alun) i fullstendig fiasko vaksine fører til alvorlige komplikasjoner ved infeksjoner i spedbarn6. Disse negative effekter av vaksinasjon kan reverseres ved en CD8+ T celle respons7. Vi har tidligere vist at de viktigste cytokiner (type jeg interferoner) brakt frem av noen stimulator av interferon gener (BRODD) agonister er avgjørende for Sertifikatklareringslisten svarene generert av disse adjuvans8, delvis ved å måle spredning OT-jeg T celler etter vaksinasjon og bruke disse treffene som et mål på CTLEN inducing evner observert i utvidet vaksinasjon tidsplaner9. Måling av spredning av OT-jeg CD8+ T celler i en vill-type (WT) C57BL/6 mottaker mus carboxyfluorescein succinimidyl ester (CFSE) fargestoff fortynning er en robust estimering av evnen til adjuvant en vaksine generere Cross-fylling av SIINFEKL, (immuno-dominante peptid av ovalbumin, OVA). Varianter av denne teknikken er mye brukt til vurdering av spredning av OT-jeg CD8+ og OT-II CD4+ T celler. For eksempel er det brukt i fravær av valgte cytokiner (KO mus) eller måle vaksinen etter antigen tilbakekalling i WT dyr. Vi utviklet en kort protokoll (4 dager eksperimentet) der etter passiv overføring av CFSE-farget OT-jeg CD8+ T celler, en subcutaneous (SC) immunisering bestående av en dose av 50 µg av endotoxin-fri OVA supplert med test adjuvans administreres (Figur 1). Oppfølging av resultatene 48 timer etter vaksinasjon gir en pålitelig bevis av kapasiteten til adjuvant til CTLEN svar. Ved denne strategien er det mulig å vurdere styrken på lokale immunrespons i drenering lymfeknute etter immunisering samt omfanget av svaret ved å måle CTL aktiviteten i milten (eller fjern lymfeknutene).

<table>
	<tbody>
		<tr>
			<td>BD LSR Fortessa Cell Analyzer</td>
			<td>BD</td>
			<td>Special Order</td>
			<td>Flow Cytometer</td>
		</tr>
		<tr>
			<td>CFSE</td>
			<td>Molecular Probes</td>
			<td>C34554</td>
			<td>Proliferation Dye</td>
		</tr>
		<tr>
			<td>MojoSort Mouse CD8 T Cell Isolation Kit</td>
			<td>Biolegend</td>
			<td>480007</td>
			<td>Magnetic Isolation Beads and antibodies for negative selection of untouched CD8 T cells.</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation</td>
			<td>Molecular Probes</td>
			<td>L23105</td>
			<td>Dead Cell Marker</td>
		</tr>
		<tr>
			<td>CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-0900-82</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>APC anti-mouse CD8a Antibody</td>
			<td>BioLegend</td>
			<td>100712</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>BV421 Rat Anti-Mouse CD4</td>
			<td>BD</td>
			<td>740007</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>Z2 coulter Particle count and Size Analyzer</td>
			<td>Beckman Coulter</td>
			<td>9914591DA</td>
			<td>Cell counter. Z2 Automated particle/cell counter</td>
		</tr>
		<tr>
			<td>EndoGrade Ovalbumin (10 mg)</td>
			<td>Hyglos(Germany)</td>
			<td>321000</td>
			<td>Ovalbumin endotoxin free tested.</td>
		</tr>
		<tr>
			<td>Cell Strainer 100&#181;m nylon</td>
			<td>Corning</td>
			<td>352360</td>
			<td>Cell strainer (100 &#181;m pore mesh cups).</td>
		</tr>
		<tr>
			<td>Sample Vials</td>
			<td>Beckman Coulter</td>
			<td>899366014</td>
			<td>Sample vials for Z2 automated counter</td>
		</tr>
		<tr>
			<td>C57BL/6 mice (CD90.2)</td>
			<td>Harlan (Rossdorf, Germany)</td>
			<td></td>
			<td>Company is now Envigo</td>
		</tr>
		<tr>
			<td>OT-I (C57BL/6 background, CD90.1)</td>
			<td>Harlan (Rossdorf, Germany)</td>
			<td></td>
			<td>Inbreed at our animal facility. Company from where adquired is now Envigo</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Fischer (Corning)</td>
			<td>14-959-5</td>
			<td>Corning Falcon Round-Bottom Polystyrene Tubes</td>
		</tr>
		<tr>
			<td>Falcon 15 mL tubes</td>
			<td>Fischer (Corning)</td>
			<td>05-527-90</td>
			<td>Falcon 15mL Conical Centrifuge Tubes</td>
		</tr>
		<tr>
			<td>PBS (500 mL)</td>
			<td>Fischer (Gibco)</td>
			<td>20-012-027</td>
			<td>Gibco PBS (Phosphate Buffered Saline), pH 7.2</td>
		</tr>
		<tr>
			<td>Red lamp (heating lamp)</td>
			<td>Dirk Rossmann GmbH (Germany)</td>
			<td>405096</td>
			<td>Heating infrred lamp (100 wats)</td>
		</tr>
		<tr>
			<td>IsoFlo (Isoflurane)</td>
			<td>Abbott Laboratories (USA)</td>
			<td>5260.04-05.</td>
			<td>Isoflurane anesthesic (250 mL flask).</td>
		</tr>
		<tr>
			<td>Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber</td>
			<td>Parkland Scientific</td>
			<td>V3000PK</td>
			<td>Isoflurane anesthesia machine.</td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>11-875-093</td>
			<td>Base medium with Glutamine (500 mL)</td>
		</tr>
		<tr>
			<td>Pen-Strept antibiotic solution (Gibco)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>15-140-148</td>
			<td>Gibco Penicillin-Streptomycin (10,000 U/mL)</td>
		</tr>
		<tr>
			<td>Fetal Bobine Serum (Gibco)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>10082147</td>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer (100 ml)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>A1049201</td>
			<td>Amonium Chloride Potasium (ACK) Whole Blood Lysis Buffer, suitable for erytrocyte lysis in spleen suspensions also</td>
		</tr>
		<tr>
			<td>Plastic Petri Dishes</td>
			<td>Nunc (distributed by ThermoFischer)</td>
			<td>150340</td>
			<td>60 x 15mm Plastic Petri Dish, Non-treated</td>
		</tr>
		<tr>
			<td>Cell Clump Filter</td>
			<td>CellTrics (Sysmex)</td>
			<td>04-004-2317</td>
			<td>CellTrics&#174; 50 &#956;m, sterile</td>
		</tr>
	</tbody>
</table>

rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development

Vurdering av moderne sub-enhet vaksiner avslører at generering av nøytralisere antistoffer er viktig, men ikke tilstrekkelig for adjuvant valg. Derfor trengs adjuvans med både humoral og cellulær immuno-stimulerende evner som kan fremme cytotoksiske T-lymfocytter (CTL) svar haster. Dermed representerer trofaste overvåking av adjuvant kandidater som induserer kryss-grunning og deretter forbedre CTL generasjon et viktig skritt i vaksineutvikling. Her presenterer vi et program for en metode som bruker SIINFEKL-spesifikke (OT-jeg) T-celler å overvåke kryss-presentasjonen av modellen antigen ovalbumin (OVA) i vivo i nærvær av forskjellige adjuvant kandidater. Denne metoden representerer en rask test for å velge adjuvans med beste kryss-grunning evner. Spredning av CD8+ T celler er den mest verdifulle indikasjonen på tvers-grunning og det anses som en relateres til adjuvant-indusert kryss-presentasjon. Denne funksjonen kan evalueres i ulike immun organer som lymfeknuter og milt. Omfanget av CTL generasjon kanne likeledes være kontrollert, og dermed gi innsikt på natur en lokal (drenering lymfeknute hovedsakelig) eller en systemisk respons (Fjern lymfeknuter og/eller milt). Denne teknikken videre kan flere modifikasjoner for testing stoffer som kan hemme bestemte kryss-presentasjon stier og tilbyr også muligheten til å bli brukt i ulike stammer konvensjonelle og genmodifiserte mus. Oppsummert vil programmet som vi presenterer her være nyttig for vaksine laboratorier i industrien eller akademia som utvikle eller modifisere kjemiske adjuvans for vaksine forskning og utvikling.

For å teste behandlinger bruker en annen kombinasjon av adjuvans (ADJ1 og ADJ2), har vi vurdert CTL generasjon kapasitet ved å måle spredning av adoptively overførte OT-jeg CD8+ T celler av flowcytometri (figur 2). For dette farget vi tidligere isolert celler fra drenering lymfeknuter og milt (tabell 1). Ved å måle spredning CD8+ T celler i lymfeknuter og milt, kunne vi bekrefte en høyere CTL generasjon kapasitet ...

Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Vi presenterer her en søknad om en standard immunologiske teknikk (CFSE farget OT-jeg spredning) skal overvåke raskt adjuvant-mediert cytotoksisk T lymfocytt (CTL) generasjon i vivo. Denne rask estimering av CTL kapasitet er nyttig for utvikling av forebyggende vaksiner mot intracellulær patogener som terapeutiske kreft vaksiner.

Rask i Vivo evaluering av adjuvants cytotoksiske T-lymfocytter generasjon evner for vaksineutvikling

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

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Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

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