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 vil gerne takke David Watkins til at understøtte de eksperimenter, gjorde det muligt for optimering af den nuværende metode. Forskning rapporteret i denne publikation blev delvist understøttet af en pilot tilskud fra Miami Center for AIDS Research af National Institutes of Health under award nummer P30AI073961. Indholdet er alene forfatternes ansvar og repræsenterer ikke nødvendigvis de officielle synspunkter National Institutes of Health.

De perifere mononukleære blodceller (PBMC) prøver anvendt i dette manuskript blev opnået fra indiske makakaber huses på Wisconsin National Primate Research Center. Disse dyr blev passet i overensstemmelse med Weatherall rapporten under en protokol godkendt af University of Wisconsin Graduate School Animal Care og brug Udvalg 33. Alle dyreforsøg blev udført under anæstesi og blev gjort alt for at minimere potentiel lidelse. 1. PKI Behandling BEMÆRK: Dette er valgfrit, men anbefales til Ma...

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Et par skridt i denne procedure fortjeneste diskussion, da de er afgørende for at give optimale resultater. Først, da kvaliteten af de biologiske prøver er en stærk indikator for succes for enhver flowcytometrisk assay 38, skal der tages alle omhu for at sikre, at cellerne er levedygtige og i suspension under farvningen procedure. Dette er særlig relevant, når der arbejdes med kryopræserverede prøver, da de er mere tilbøjelige til at klumpe og indeholder typisk højere antal døde celler. I disse til...

CD8 + T-celler omfatter en afgørende komponent i det adaptive immunsystem, som de deltager i tumor immun overvågning og bidrage til udryddelse af intracellulære patogener 1. I enkle vendinger, CD8 + T-celler udtrykker T-cellereceptorer (TCR&#39;er), som specifikt genkender peptid-dominerende histokompatibilitetskompleks klasse I (pMHC-I) molekyler til stede på plasmamembranen af værtscellerne. Da disse peptider er afledt af proteolyse af endogent syntetiserede proteiner, celleoverflade pMHC-I-komplekser give et vindue ind i intracellulære miljø. Ved virusinfektion, for eksempel, vil inficerede celler udviser MHC-I-molekyler, der indeholder virus-afledte peptider, der kan tjene som ligander for TCR udtrykt af patruljerer CD8 + T-celler. I tilfælde en virus-specifikke CD8 + T-celle møder en inficeret celle præsenterer sin pMHC-I-ligand, vil TCR engagement resultere i CD8-aktivering + T-celle og Ultiende føre til at målrette cellelyse. I betragtning af den kritiske karakter af disse TCR / pMHC-I-interaktioner, bestemmelse af størrelsen, specificitet og fænotype for at reagere CD8 + -T-celler kan ofte afsløre vigtige fingerpeg om humane sygdomme. Indtil begyndelsen af 1990&#39;erne, kvantificering af Ag-specifikke CD8 + T-celler har påberåbt sig den teknisk krævende begrænsende fortynding assay (LDA) 2,3. Ikke kun gjorde LDA kræve flere dage at være afsluttet, er det heller ikke at opdage celler, der manglede proliferative potentiale. Som et resultat heraf LDA voldsomt undervurderet den faktiske frekvens af antigen (Ag) -specifikke CD8 + -T-celler, der deltager i en immunreaktion. Selv om udviklingen af ELISPOT og intracellulære cytokin farvende assays stærkt forbedret evnen til at måle cellulær immunitet, disse metoder stadig påkrævet in vitro-stimulering til kvantificering Ag-specifikke T-celler 4. Det var ikke før 1996, at Altman, Davis, og colleagues offentliggjort deres skelsættende artikel rapporterer udviklingen af pMHC-I tetramer teknologi 5. Afgørende for succes af denne teknik var den multimerisering af pMHC-I-molekyler, der forlængede halveringstiden af ​​TCR / pMHC-I-interaktioner, og derved reducere sandsynligheden for pMHC-I tetramerer falde ned under vaske- trinnene flowcytometriske assays. Den største fordel ved pMHC-I tetramerer forhold til de ovennævnte assays er evnen til nøjagtigt at detektere Ag-specifikke CD8 + T-celler direkte ex vivo uden behov for in vitro restimulering. Desuden er en kombination af pMHC-I tetramerfarvning med flerfarvede flowcytometri har tilladt detaljerede analyser af differentieringstrinet, hukommelse fænotype, og aktiveringsstatus af Ag-specifikke CD8 + T-celler 2-4. I lyset af de seneste tekniske fremskridt til karakterisering CD8 + T-celle repertoirer af pMHC-I multimer farvning 6, bredden af applikationer for denne metode vil sandsynligvis fortsætte med at udvide. Kun få områder i biomedicinsk forskning har nydt mere fra pMHC-I tetramerfarvning end inden for HIV immunologi 7. Selvom CD8 + T-celler var blevet tidsmæssigt tilknyttet det oprindelige kontrol af HIV viræmi på tidspunktet for offentliggørelsen af Altman, Davis, og kolleger 8,9, brug af pMHC-I tetramerer i de følgende år væsentligt udvidet vores forståelse af HIV-specifikke CD8 + T-celle-respons. For eksempel pMHC-I tetramerfarvning bidrage til at bekræfte den robuste størrelse virus-specifikke CD8 + T-celle-responser i de fleste HIV-inficerede individer 10-12. Denne metode lettede også karakteriseringen af HIV og SIV-specifikke CD8 + T-celle-responser begrænset af MHC-I-molekyler associeret med spontan kontrol af viral replikation i fravær af antiretroviral behandling-et fænomen kendt som &quot;elite kontrol&quot; <sup&gt; 13-15. Endvidere pMHC-I tetramerer var med til at etablere den programmerede død 1 (PD-1) / PD ligand 1 (PD-L1) akse som en reversibel pathway for dysfunktionel fænotype af HIV-specifikke CD8 + T-celler i ukontrolleret kronisk infektion 16,17. Kollektivt, disse undersøgelser understreger anvendeligheden af pMHC-I tetramerer til overvågning CD8 + T-cellereaktioner mod AIDS-virus. Eksperimentel SIV-infektion af rhesus makakaber (Macaca mulatta) er fortsat den bedste dyremodel for at evaluere immune indgreb mod hiv / aids 18,19. I de seneste 25 år er der sket betydelige fremskridt i identifikation og karakterisering af SIV-specifikke CD8 + T-celler i denne abearter, herunder opdagelsen af MHC-I alleler og definitionen af peptid bindende motiver 13,20-27 . Som følge heraf har pMHC-I tetramerer blevet udviklet til analyse af SIV-specifikke CD8 + T-cellereaktioner i denne dyremodel 28. De fleste af disse reagenser er lavet af genprodukterne af fire rhesus makak MHC-I-alleler: Mamu-A1 * 001, Mamu-A1 * 002: 01, Mamu-B * 008: 01, og Mamu-B * 017: 01. Notatet gør rhesus makakaber ikke udtrykke en MHC-C locus 29. Langt størstedelen af ​​de pMHC-I tetramerer anvendt i de foreliggende eksperimenter blev produceret hos NIH Tetramer Core Facility fra Emory University. Ikke desto mindre er nogle af disse reagenser, herunder Mamu-A1 * 001 tetramerer bundet til immundominerende Gag CM9 epitop, kan kun opnås fra kommercielle kilder på grund af licensaftaler. Brug af pMHC-I tetramerer af de fire rhesus macaque alleler anført ovenfor, har vi med succes opregnet CD8 + T-celler mod i alt 21 SIV epitoper (tabel 1), som blev induceret ved vaccination eller primær SIV infektion 30,31 (Martins et al., upublicerede observationer). Den foreliggende manuskript tilvejebringer enoptimeret pMHC-I tetramerfarvning protokol til bestemmelse af frekvensen og hukommelse fænotypen af SIV-specifikke CD8 + T-celler i makakaber. Assayet begynder med en elektiv 30 minutters inkubation med et protein kinase inhibitor (PKI, her er Dasatinib anvendt) for at reducere TCR-internalisering og derved forbedre pMHC-I tetramerfarvning 32. Som beskrevet nedenfor, denne behandling er især nyttig, når du bruger Mamu-B * 017: 01 tetramerer. Instruktioner om, hvordan til at mærke cellerne med fluorokromkonjugerede pMHC-I tetramerer og monoklonale antistoffer (MAB) findes også. Denne protokol omfatter også en celle permeabilisering trin for den intracellulære detektion af cytolyse-associerede molekyle granzym B (Gzm B). MAb mod CD3 tilsættes ved dette trin, samt at forbedre påvisningen af ​​denne TCR signalmolekyle. Som reference er alle fluorochromer anvendes i denne farvning panel angivet.

<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-dominerende histokompatibilitetskompleks klasse I (pMHC-I) tetramerer har været et uvurderligt værktøj til at studere CD8 + T-cellereaktioner. Fordi disse reagenser direkte binde til T-celle-receptorer på overfladen af CD8 + T-lymfocytter, fluorochrommærkede pMHC-I tetramerer muliggøre nøjagtig påvisning af antigen (Ag) -specifik CD8 + T-celler uden behov for in vitro-re -stimulation. Desuden, når kombineret med flerfarvede flowcytometri, kan pMHC-I tetramerfarvning afsløre vigtige aspekter af Ag-specifikke CD8 + T-celler, herunder differentiering fase, hukommelse fænotype, og aktiveringsstatus. Disse typer af analyser har været særligt anvendelige inden for HIV immunologi, hvor CD8 + T-celler kan påvirke progression til AIDS. Eksperimentel infektion af rhesus-makakaber med abeimmundefektvirus (SIV) tilvejebringer et uvurderligt værktøj til at studere cellulær immunitet mod AIDS-virus. Som følge heraf betydelig PROGREss er gjort med at definere og karakterisere T-celle responser i denne dyremodel. Vi præsenterer her en optimeret protokol for optælling SIV-specifikke CD8 + T-celler i makakaber efter pMHC-I tetramerfarvning. Vores assay tillader den samtidige kvantificering og hukommelse fænotypebestemmelse af to pMHC-I tetramer + CD8 + T-cellepopulationer pr test, som kan være nyttig til at spore SIV-specifikke CD8 + T-celle-responser frembragt af vaccination eller SIV-infektion. I betragtning af relevansen af ikke-humane primater inden for biomedicinsk forskning, denne metode kan anvendes til at studere CD8 + T-celle responser i flere indstillinger sygdom.

Den her beskrevne protokol er blevet anvendt til at bestemme størrelsen og hukommelse fænotypen af vaccineinducerede, Gag CM9-specifikke CD8 + T-cellereaktioner i en Mamu-A1 * 001 + rhesus makak. Til denne analyse blev et APC-konjugeret Mamu-A1 * 001 / Gag CM9 tetramer anvendes i en 8-farve flowcytometrisk farvning panel. Figur 1A - F viser gating anvendte strategi til at analysere de data, som skal anvendes for begge tetramerer til...

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 præsenteres en optimeret protokol til optælle og karakterisering af rhesus makak CD8 + T-celler mod AIDS-virus. Denne artikel er ikke kun nyttig til området for HIV immunologi, men også til andre områder af biomedicinsk forskning, hvor CD8 + T-cellereaktioner er kendt for at påvirke udfaldet sygdom.

Analyse af Simian Immunodeficiency Virus-specifikke CD8<sup&gt; +</sup&gt; T-celler i makakaber efter Peptid-MHC-I tetramerfarvning

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

Bestemmelse Optimal cytotoksisk aktivitet for Human Her2neu Specifikke CD8 T-celler ved at sammenligne de Cr51 Udgivelse Assay 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 ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Ændring af primære humane T-celler

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

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

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 ...

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

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

Induktion af murine tarmbetændelse ved adoptiv overførsel af effektor CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Høj</sup&gt; T celler i immunsvækkede mus

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

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

Fremstilling af CD4<sup&gt; +</sup&gt; T-celler til analyse af GD3 og GD2 gangliosidet Membrane 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 farvning og kvantitativ analyse til at bestemme placering, overflod og fænotype af Antigen-specifikke CD8 T celler i væv

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

Zika Virus Specific Diagnostic Epitope Discovery

Zika Virus specifikke diagnostiske epitop opdagelse

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques