Name: analysis of simian immunodeficiency virus-specific cd8+ t-cells in rhesus macaques by peptide-mhc-i tetramer staining
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Description: Watch this Scientific Journal Video about Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining at JoVE.com

Vi ønsker å takke David Watkins for å støtte forsøkene som gjorde det mulig optimalisering av dagens metodikk. Forskning rapportert i denne publikasjonen ble støttet delvis av en pilot stipend gitt av Miami Senter for AIDS forskning av National Institutes of Health i henhold award nummer P30AI073961. Innholdet er utelukkende ansvaret til forfatterne og ikke nødvendigvis representerer den offisielle visninger av National Institutes of Health.

De perifert blod mononukleære celle (PBMC) prøver benyttes i dette manuskriptet ble hentet fra indiske rhesusape plassert på Wisconsin National Primate Research Center. Disse dyrene var ivaretatt i samsvar med Weatherall Rapporter under en protokoll godkjent av University of Wisconsin Graduate School Animal Care og bruk komité 33. Alle dyr prosedyrer ble utført under anestesi og alle anstrengelser ble gjort for å minimere potensiell lidelse. 1. PKI Treatment MERK: Dette er valgfritt, men a...

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Noen få skritt i denne prosedyren fortjeneste diskusjonen som de er avgjørende for ettergivende optimale resultater. Først, siden kvaliteten av de biologiske prøver er en sterk prediktor for suksess for enhver flowcytometrisk analyse 38, alt forsiktighet må utvises for å sikre at cellene er levedyktige og i suspensjon i løpet av fargeprosedyre. Dette er særlig aktuelt når man arbeider med cryopreserved prøvene siden de er mer tilbøyelig til klumpdannelse og inneholder typisk høyere antall døde ce...

CD8 + T-celler utgjør en viktig del av det adaptive immunsystemet som de deltar i tumor immunovervåkning og bidra til utryddelse av intracellulære patogener 1. Enkelt sagt, CD8 + T-celler som uttrykker T-celle-reseptorer (TCR) som spesifikt gjenkjenner peptid-store histokompatibilitetskompleks klasse I (pMHC-I) molekyler tilstede på plasmamembranen til vertsceller. Siden disse peptidene er avledet fra proteolyse av endogent syntetiserte proteiner, celleoverflate pMHC-I komplekser tilveiebringe et vindu inn i det intracellulære miljøet. Ved virusinfeksjon, for eksempel, vil infiserte celler viser MHC-I molekyler inneholdende virus-avledede peptider som kan tjene som ligander for TCR uttrykt av patruljerende CD8 + T-celler. I tilfelle en virus-spesifikke CD8 + T-celle møter en infisert celle som presenterer sin pMHC-I-ligand, vil TCR inngrep resultere i CD8 + T-celleaktivering og ultiinstans føre til målet cellelyse. Gitt den kritiske naturen av disse TCR / pMHC-I interaksjoner, bestemme omfanget, spesifisitet, og fenotype reagere CD8 + T-celler kan ofte avdekke viktige ledetråder om menneskelige sykdommer. Fram til tidlig på 1990-tallet, kvantifisering av Ag-spesifikke CD8 + T-celler er avhengige av teknisk krevende begrensende fortynning analysen (LDA) 2,3. Ikke bare gjorde LDA kreve flere dager å bli ferdig, det også klart å påvise celler som manglet proliferativ potensial. Som et resultat av LDA vesentlig underestimert den faktiske frekvensen av antigen (Ag)-spesifikke CD8 + T-celler som deltar i en immunrespons. Selv om utviklingen av ELISPOT og intracellulære cytokin fargings assays sterkt forbedret evne til å måle cellulær immunitet, disse fremgangsmåtene fremdeles nødvendig in vitro stimulering for kvantifisering av Ag-spesifikke T-celler 4. Det var ikke før 1996 at Altman, Davis, og colleagues publisert deres landemerke artikkelen rapporterer utviklingen av pMHC-I tetramer teknologi 5. Kritisk for å lykkes med denne teknikken ble den multimerization av pMHC-I-molekyler, som forlenget halveringstid på TCR / pMHC-I interaksjoner, for derved å redusere sannsynligheten for pMHC-I tetramerer faller av under vasketrinnene av strømningscytometriske analyser. Den største fordelen med pMHC-I tetramerer enn de ovennevnte analysene er muligheten for nøyaktig bestemmelse av Ag-spesifikke CD8 + T-celler direkte ex vivo uten behov for in vitro re-stimulering. Videre er kombinasjonen av pMHC-I tetramer farving med flere farge flowcytometri har tillatt detaljerte analyser av differensieringstrinnet, hukommelse fenotype, og aktiveringsstatusen til Ag-spesifikke CD8 + T-celler 2-4. I lys av de siste tekniske fremskritt for karakterisering av CD8 + T-celle repertoar av pMHC-I multimer flekker 6, bredden av applikasjoner for denne metoden vil trolig fortsette å utvide. Få områder i biomedisinsk forskning har dratt nytte mer fra pMHC-I tetramer flekker enn innen hiv immunologi 7. Selv om CD8 + T-celler var blitt temporalt forbundet med den første kontrollen av HIV-viremi ved tidspunktet for den publikasjon av Altman, Davis og medarbeidere 8,9, bruk av pMHC-I tetramerer i de påfølgende år utvidet vår forståelse av betydelig HIV-spesifikke CD8 + T-cellerespons. For eksempel, pMHC-I tetramer farging bidratt til å bekrefte den sterke størrelsen av virus-spesifikke CD8 + T-celle-responser i de fleste HIV-infiserte individer 10-12. Denne metodikken også til rette for karakterisering av HIV- og SIV-spesifikke CD8 + T-celle-responser begrenses av MHC-I molekyler forbundet med spontan kontroll av viral replikasjon i fravær av antiretroviral behandling-et fenomen kjent som &quot;elite kontroll&quot; <sup&gt; 13-15. Videre pMHC-I tetramers var instrumental i å etablere programmert død 1 (PD-1) / PD ligand 1 (PD-L1) aksen som en reversibel vei for dysfunksjonelle fenotype av HIV-spesifikke CD8 + T-celler i ukontrollert kronisk infeksjon 16,17. Sammen er disse studier underanvendeligheten av pMHC-I tetramerer for overvåkning av CD8 + T-celle responser mot AIDS-virus. Eksperimentell SIV infeksjon av rhesusape (Macaca Mulatta) er fortsatt den beste dyremodell for å vurdere immun intervensjoner mot HIV / AIDS 18,19. I de siste 25 årene har store fremskritt er gjort i identifisering og karakterisering av SIV spesifikke CD8 + T-celler i denne apearter, inkludert oppdagelsen av MHC-I alleler og definisjonen av peptid binding motiver 13,20-27 . Som et resultat, har pMHC-I-tetramerer er utviklet for analyse av SIV-spesifikke CD8 + T-cellesvarene i denne dyremodell 28. De fleste av disse reagenser er laget av genprodukter av fire rhesusape MHC-I-alleler: Mamu-A1 * 001, Mamu-A1 * 002: 01, Mamu-B * 008: 01, og Mamu-B * 017: 01. Av notatet, rhesusape ikke uttrykke en MHC-C locus 29. De aller fleste av de pMHC-I tetramers som brukes i dagens eksperimenter ble produsert ved NIH Tetramer Kjerne Facility ved Emory University. Likevel, noen av disse reagenser, inkludert Mamu-A1 * 001 tetramers bundet til immunodominant Gag CM9 epitop, kan kun fås fra kommersielle kilder på grunn av lisensavtaler. Bruke pMHC-I tetramers av de fire rhesusape alleler som er nevnt ovenfor, har vi lykkes nummerert CD8 + T-celler mot totalt 21 SIV epitoper (tabell 1), som ble utløst av vaksinasjon eller primær SIV infeksjon 30,31 (Martins et al., upubliserte observasjoner). Den nåværende manuskriptet gir enoptimalisert pMHC-I tetramer farging protokoll for å bestemme frekvensen og hukommelse fenotype av SIV-spesifikke CD8 + T-celler i rhesusape. Analysen starter med et valg 30 min inkubering med en protein kinase inhibitor (PKI, her blir Dasatinibs anvendes) for å redusere TCR internalisering og derved forbedre pMHC-I tetramer farging 32. Som beskrevet nedenfor, er denne behandlingen spesielt nyttig når du bruker Mamu-B * 017: 01 tetramers. Instruksjoner om hvordan du merke celler med fluorokromkonjugerte konjugert pMHC-I tetramers og monoklonale antistoffer (mAbs) tilbys også. Denne protokollen innbefatter også en celle permeabiliseringen trinn for den intracellulære påvisning av cytolyse-assosiert molekyl granzyme B (Gzm B). MAb mot CD3 tilsettes ved dette trinnet i tillegg til å forbedre deteksjon av denne TCR signalmolekyl. Som en referanse, er alle fluorokromer anvendes i denne fargingen panel oppført.

<table border="0" cellpadding="0" cellspacing="0" width="1449">
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			<td height="21" style="height:21px;width:329px;">Dasatinib</td>
			<td style="width:236px;">Axon medchem</td>
			<td style="width:127px;">Axon&#160;1392</td>
			<td style="width:757px;">Must be resuspended in DMSO and immediately stored at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI w/ Glutamax</td>
			<td>gibco/ Life Technologies</td>
			<td>61870-036</td>
			<td>Must be stored at 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat Inactivated FBS</td>
			<td>gibco/ Life Technologies</td>
			<td>10082-147</td>
			<td>Must be stored at 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycinp-Amphotericin B</td>
			<td>Lonza</td>
			<td>17-745E</td>
			<td>Must be stored at 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO, Anhydrous</td>
			<td>Life Technologies</td>
			<td>D12345</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-mL Round-Bottom Polypropylene Tubes</td>
			<td>VWR</td>
			<td>60819-728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorochrome-conjugated pMHC-I tetramers</td>
			<td>NIH Tetramer Core or MBL, Inc.</td>
			<td></td>
			<td>Must be stored and maintained at&#160; 4 &#176;C. Centrifuge at 20,000 x g for 15 min before use. Do not freeze.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorochrome-conjugated mAbs</td>
			<td>Various companies</td>
			<td></td>
			<td>Must be stored and maintained at&#160; 4 &#176;C. Do not freeze.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD&#160;Fixable Aqua&#160;Dead&#160;Cell Stain Kit</td>
			<td>Life Technologies</td>
			<td>l34957</td>
			<td>Must be stored at -20 &#176;C. Resuspend each aliquot in 50 &#956;L of DMSO prior to use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brilliant Stain Buffer</td>
			<td>BD Biosciences</td>
			<td>563794</td>
			<td>Must be stored at&#160; 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline</td>
			<td>VWR</td>
			<td>97064-158</td>
			<td>Store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumine Bovine</td>
			<td>VWR</td>
			<td>700011-230</td>
			<td>Must be stored at&#160; 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Azide</td>
			<td>VWR</td>
			<td>97064-646</td>
			<td>Store at room temperature. Toxic substance. Do not mix with bleach.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bleach</td>
			<td>VWR</td>
			<td>89501-620</td>
			<td>Corrosive chemical, cannot be mixed with sodium azide. Handle with care</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714-S</td>
			<td>Flammable, corrosive, and toxic reagent. Handle with care</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polysorbate 20 (Tween-20)</td>
			<td>Alfa Aesar</td>
			<td>L15029</td>
			<td>Store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permeabilization Solution 2&#160;</td>
			<td>BD Biosciences</td>
			<td>340973</td>
			<td>Toxic and corrosive reagent. Handle with care</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sarsdet Tubes 1.5mL screw top</td>
			<td>VWR</td>
			<td>72.692.005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.0ml DNA/RNA Low bind Tubes</td>
			<td>Eppendorf</td>
			<td>22431048</td>
			<td>The use of Sterile microtubes is preffered&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex mixer</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biosafety Cabinet&#160;</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Milli Q Intergral Water Purification system</td>
			<td>EMD Millipore</td>
			<td>ZRXQ010WW</td>
			<td>Molecular Biology grade water from any provider may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4 &#176;C&#160; refrigerator</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD LSR II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer must contain lasers and filters that are compatible with the staining panel used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deionized water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum Foil</td>
			<td>VWR SCIENTIFIC INC.</td>
			<td>89068-738</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td></td>
			<td></td>
			<td>Must be able to maintain 37 &#176;C&#160; internal temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS Diva software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flowjo software version 9.6</td>
			<td>Flowjo</td>
			<td></td>
			<td>Used to analyze FCS files generated by FACS Diva software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropippette tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of simian immunodeficiency virus-specific cd8+ t-cells in rhesus macaques by peptide-mhc-i tetramer staining

Peptid-store histokompatibilitetskompleks klasse I (pMHC-I) tetramerer har vært et uvurderlig verktøy for å studere CD8 + T-celleresponser. Fordi disse reagensene direkte binder seg til T-celle-reseptorer på overflaten av CD8 + T-lymfocytter, fluorokromkonjugerte merket pMHC-I tetramerer muliggjøre nøyaktig deteksjon av antigen (Ag) -spesifikk CD8 + T-celler uten behov for in vitro gjen -stimulering. Dessuten, når det kombineres med flere farge flowcytometri, kan pMHC-I tetramer farging åpenbare nøkkelaspekter ved Ag-spesifikke CD8 + T-celler, innbefattende differensieringstrinn, hukommelse fenotype, og aktiveringsstatusen. Slike analyser er særlig nyttig på området HIV immunologi hvor CD8 + T-celler kan påvirke progresjon til AIDS. Eksperimentell infeksjon av rhesusape med Simian immunsviktvirus (SIV) gir et uvurderlig verktøy for å studere cellulær immunitet mot AIDS-viruset. Som et resultat av betydelig progress har blitt gjort i å definere og karakterisere T-celleresponser i denne dyremodell. Her presenterer vi en optimalisert protokoll for opplisting SIV spesifikke CD8 + T-celler i rhesusape av pMHC-I tetramer farging. Vår analyse tillater den samtidige kvantifisering og hukommelse fenotyping av to pMHC-I tetramer + CD8 + T-celle-populasjoner per test, som kan være nyttig for sporing av SIV-spesifikke CD8 + T-celle-responser generert ved vaksinering eller SIV-infeksjon. Med tanke på relevansen av ikke-humane primater i biomedisinsk forskning, er denne metodikk anvendbar for å studere CD8 + T-celleresponser i flere sykdoms innstillinger.

Protokollen er beskrevet her har blitt brukt for å bestemme størrelsen og hukommelse fenotype av vaksineinduserte, Gag CM9-spesifikke CD8 + T-celleresponser i et Mamu-A1 * 001 + rhesusape. For denne analysen ble en APC-konjugert Mamu-A1 * 001 / Gag CM9 tetramer brukes i en 8-farge flowcytometrisk flekker panel. Figur 1A - F viser gating strategien brukes for å analysere dataene som skal brukes for både tetramers stede i hver test....

Watch this Scientific Journal Video about Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining at JoVE.com

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Her presenterer vi en optimalisert protokoll for opplisting og karakter rhesusape CD8 + T-celler mot AIDS-viruset. Denne artikkelen er nyttig ikke bare til feltet av HIV immunologi, men også til andre områder av biomedisinsk forskning hvor CD8 + -T-celleresponser er kjent for å påvirke sykdomsforløp.

Analyse av Simian Immunodeficiency Virus-spesifikke CD8<sup&gt; +</sup&gt; T-celler i rhesusape av Peptide-MHC-I Tetramer Farging

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

Research

JoVE Journal

Immunology and Infection

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Bestemme Optimal cytotoksisk aktivitet for Human Her2neu Spesifikke CD8 T-celler ved å sammenligne Cr51 Slipp analysen til xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

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The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

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A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

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Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

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There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

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Utarbeidelse av CD4<sup&gt; +</sup&gt; T celler for analyse av GD3 og GD2 Gangliosid Membran Expression ved mikroskopi

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

In Situ MHC-tetramer flekker og kvantitativ analyse for å fastslå plasseringen, overflod og fenotypen av Antigen-spesifikke CD8 T celler i vev

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