Name: in vivo assay for detection of antigen-specific t-cell cytolytic function using a vaccination model
Uploaded: 2017-11-28T12:30:00
Description: Watch this Scientific Journal Video about In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model at JoVE.com

Dette arbejde støttes af NIH forskning (A1R03AI120027 (RN) og 1R21AI20012 (RN)), institutionelle forskning tilskud (RN), start-up tilskud (RN), og MD Anderson CIC frø yde (RN).

Alle metoder beskrevet her er blevet godkendt af institutionelle Animal Care og brug udvalg (IACUC) på University of Texas MD Anderson Cancer Center.
1. forberedelse af peptid for vaccinen
<ol><li>Opløse den frysetørrede peptid med fosfatbufferet saltopløsning (PBS) til den passende koncentration og vortex for 30 s. Bemærk: For hgp10025-33, endelige koncentration er 1 mg/mL og for æg257-264, den endelige koncentration...

<ol>
	<li>Liu, L., 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=Visualization+and+quantification+of+T+cell-mediated+cytotoxicity+using+cell-permeable+fluorogenic+caspase+substrates.">Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates.</a> Nat Med. 8 (2), 185-189 (2002).</li><li>Wherry, E. J., 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=Lineage+relationship+and+protective+immunity+of+memory+CD8+T+cell+subsets.">Lineage relationship and protective immunity of memory CD8 T cell subsets.</a> Nat Immunol. 4 (3), 225-234 (2003).</li><li>He, L., 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=A+sensitive+flow+cytometry-based+cytotoxic+T-lymphocyte+assay+through+detection+of+cleaved+caspase+3+in+target+cells.">A sensitive flow cytometry-based cytotoxic T-lymphocyte assay through detection of cleaved caspase 3 in target cells.</a> J Immunol Methods. 304 (1-2), 43-59 (2005).</li><li>Barber, D. L., Wherry, E. J., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+rapid+in+vivo+killing+by+memory+CD8+T+cells.">Cutting edge: rapid in vivo killing by memory CD8 T cells.</a> J Immunol. 171 (1), 27-31 (2003).</li><li>Quah, B. J., Wijesundara, D. K., Ranasinghe, C., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fluorescent+target+arrays+for+assessment+of+T+cell+responses+in+vivo.">The use of fluorescent target arrays for assessment of T cell responses in vivo.</a> J Vis Exp. (88), e51627 (2014).</li><li>Johansson, 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=Natural+killer+cell+education+in+mice+with+single+or+multiple+major+histocompatibility+complex+class+I+molecules.">Natural killer cell education in mice with single or multiple major histocompatibility complex class I molecules.</a> J Exp Med. 201 (7), 1145-1155 (2005).</li><li>Hailemichael, Y., 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=Persistent+antigen+at+vaccination+sites+induces+tumor-specific+CD8(+)+T+cell+sequestration,+dysfunction+and+deletion.">Persistent antigen at vaccination sites induces tumor-specific CD8(+) T cell sequestration, dysfunction and deletion.</a> Nat Med. 19 (4), 465-472 (2013).</li><li>Overwijk, W. W., 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=Tumor+regression+and+autoimmunity+after+reversal+of+a+functionally+tolerant+state+of+self-reactive+CD8++T+cells.">Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells.</a> J Exp Med. 198 (4), 569-580 (2003).</li><li>Cho, H. I., Celis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+peptide+vaccines+eliciting+extensive+CD8+T-cell+responses+with+therapeutic+antitumor+effects.">Optimized peptide vaccines eliciting extensive CD8 T-cell responses with therapeutic antitumor effects.</a> Cancer Res. 69 (23), 9012-9019 (2009).</li><li>Ya, Z., Hailemichael, Y., Overwijk, W., Restifo, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+for+pre-clinical+study+of+human+cancer+immunotherapy.">Mouse model for pre-clinical study of human cancer immunotherapy.</a> Curr Protoc Immunol. 108, 21-43 (2015).</li><li>Menon, V., Thomas, R., Ghale, A. R., Reinhard, C., Pruszak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+protocols+for+surface+and+intracellular+antigen+analyses+of+neural+cell+types.">Flow cytometry protocols for surface and intracellular antigen analyses of neural cell types.</a> J Vis Exp. (94), (2014).</li><li>Durward, M., Harms, J., Splitter, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+specific+killing+assay+using+CFSE+labeled+target+cells.">Antigen specific killing assay using CFSE labeled target cells.</a> J Vis Exp. (45), (2010).</li></ol>

Denne protokol er ligetil, er der et par kritiske trin, der skal udføres omhyggeligt. Covax priming efter injektion af antigen-specifikke T-celle bliver testet er nødvendige for at se alle drab på de pulserende mål. Mens det er muligt at vandbaseret covax vaccination skaber en akut inflammatorisk tilstand, for de kronisk inflammatoriske fase, kan erstatter den kortlivede vand-baseret formulering med en langsom-antigen release olie-baserede tilgang producere en bedre resultat7,<sup c...

Flere protokoller findes til at vurdere cytolytisk (CTL) af en CD8+ eller CD4+ T-celle. Denne vurdering kan let gøres i vitro under kontrollerede forhold1,2,3. Derudover infektionssygdom modeller, såsom LCMV, klassisk har undersøgt CTL funktion ved hjælp af varierende CFSE (5-(and 6)-Carboxyfluorescein diacetat succinimidyl ester) mærket målceller hvor CFSEHej-mærket celler er pulserende med en peptid og CFSElo-mærket målet celler er tilbage unpulsed. Cellerne er derefter sprøjtes i forholdet 1:1 og vurderet for tabet af CFSEHej-mærket pulserende mål af flow flowcytometri4. Vaccine og afvisning modeller har også brugt lignende strategier til vurdering af, i vivo drab af begge CD8+ og CD4+ T-celler såvel som NK celler5,6. Dette er en kraftfuld assay, men kræver brug af smitstoffer, der prime immunsystemet inden målet injektionen.
Denne protokol, på den anden side kræver ingen forudgående infektion af værten og i stedet udnytter en vaccinationsstrategi for at prime immunsystemet inden målet injektionen. Denne vaccination består af en vand-baseret formulering af peptid vaccine, som kræver tilvejebringelse af en immunstimulerende cocktail kaldet covax7, bestående af en Toll-lignende receptor 7 (TLR7) agonist (imiquimod creme), en agonistisk anti-CD40 antistof, og interleukin-2 (IL-2) fører til synergistisk kombination af immunstimulerende agenter for udvikling af peptid-specifikke priming og robust immunrespons. Som sådan, giver denne analyse en hurtig udlæsning af listen over Tillidscertifikater funktion som vaccinen er indgive sammen med celler for vurdering af funktion kun tre dage før injektion af target-cellerne. Derudover er covax priming stærke nok, at drab kapacitet af PRIMES antigen-specifikke T-celle kan ses mellem 4 og 24 timer efter injektion.
De store begrænsning af denne protokol er at identificere tidslinjen i vivo til påvisning af target drab, der er egnet til både antigenet og den hypotese at være testet. Omhyggelig vurdering skal udføres, som variationer i antigen styrke samt genetiske ændringer bliver testet i T-celler kan resultere i differential CTL-funktion, som ville kræve en forskellig timing påvisning af target drab. Hertil kommer, mens den passende koncentration af peptid for vaccination er blevet optimeret til menneskelige melanom antigen glycopeptidgruppen 100 (hgp10025-33) og ægalbumin257-264 (OVA257-264)8,9 , brug af en anden antigen model, der kan være mere hensigtsmæssigt at en given undersøgelse ville kræve yderligere validering. På grund af forventede forskelle i en mål antigener evne til at stimulere CTL effektor funktion i kombination med covax som en adjuvant, kan optimering af IL-2 dosis koncentration og dosis frekvens være afgørende for at nå de ønskede mål. Samlet set denne protokol giver mulighed for en hurtig vurdering af dræbe funktion i vivo og kan tilpasses til enhver given antigen.

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			<td height="20" style="width:199pt;height:15.0pt" width="265">6- to 12-week old female C57BL/6 mice&#160;</td>
			<td class="xl69" style="width:110pt" width="146">Charles River</td>
			<td class="xl70" style="width:89pt" width="119">027</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">OT-1 6-12-week old female mice&#160;</td>
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			<td height="24" style="height:18.0pt">hgp10025&#8211;33&#160;</td>
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			<td height="20" style="height:15.0pt">70% isopropyl alcohol Prep&#160;</td>
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			<td height="20" style="height:15.0pt">PBS</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">FBS</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">RBC lysis buffer&#160;</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">RPMI 1640 Media</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">11875119</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">L-Glutamine&#160;</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">25030081</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">Pen/Strep</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">15140122</td>
			<td>penicillin/streptomycin</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">CFSE</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">C34554</td>
			<td>5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">Bovine serum albumin (BSA)</td>
			<td class="xl70" style="width:110pt" width="146">Sigma&#160;</td>
			<td class="xl70" style="width:89pt" width="119">A4503</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">1.5 mL MCT graduated natural&#160;</td>
			<td class="xl70" style="width:110pt" width="146">Fisher</td>
			<td class="xl70" style="width:89pt" width="119">05-408-129</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">70% ethanol</td>
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			<td class="xl73">BP8201500</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">Trypan blue solution, 0.4%&#160;&#160;</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">Hemocytometer</td>
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			<td class="xl70" style="width:89pt" width="119">267110</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">27 gauge needle</td>
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			<td class="xl70" style="width:89pt" width="119">305109</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">1 mL syringe</td>
			<td class="xl70" style="width:110pt" width="146">BD</td>
			<td class="xl70" style="width:89pt" width="119">309659</td>
			<td></td>
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in vivo assay for detection of antigen-specific t-cell cytolytic function using a vaccination model

Nuværende metoder til antigen-specifikke drab er begrænset til in vitro- brug eller udnyttet i infektionssygdom modeller. Der er imidlertid ikke en protokol, der er specielt beregnet til at måle antigen-specifikke drab uden en infektion. Denne protokol er designet og beskriver metoder til at overvinde disse begrænsninger ved at tillade til påvisning af antigen-specifikke drab af en målcelle ved CD8+ T-celler i vivo. Dette opnås ved at flette en vaccination model med en traditionel CFSE-mærket målet dræbe assay. Denne kombination gør det muligt for forskeren at vurdere antigen-specifikke CTL potentiale direkte og hurtigt som analysen ikke er afhængig af tumorvækst eller infektion. Derudover udlæsningen er baseret på flowcytometri og så skal være let tilgængelige for de fleste forskere. De store begrænsning af undersøgelsen er at identificere den tidslinjen i vivo , der passer til den hypotese at være testet. Variationer i antigen styrke og mutationer i T-celler, der kan resultere i differential cytolytisk funktion skal vurderes omhyggeligt for at fastslå det optimale tidspunkt for celle høst og vurdering. Den passende koncentration af peptid for vaccination er blevet optimeret til hgp10025-33 og æg257-264, men yderligere validering vil være behov for andre peptider, der kan være mere hensigtsmæssigt at en given undersøgelse. Samlet set denne protokol giver mulighed for en hurtig vurdering af dræbe funktion i vivo og kan tilpasses til enhver given antigen.

Inden injektion af CFSE-mærket target-cellerne køres 1:1 celle blandingen på en flow forskellige til at bestemme baseline frekvenser af både den CFSEHej og CFSElo target-cellerne. Figur 1 A viser den gating strategi til at opdage ændringer i CFSE populationer, en indledende gate er lavet ved hjælp af FSC og SSC parametre. De samlede CFSE-positive celler er derefter subgated forud for vurdering af ændringer i hyppi...

Watch this Scientific Journal Video about In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model at JoVE.com

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Målet med denne protokol er at give mulighed for detektion af in vivo antigen-specifikke drab af en målcelle i en murine model.

In Vivo Assay til påvisning af Antigen-specifikke T-celle cytolytisk funktion ved hjælp af en Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Research

JoVE Journal

Immunology and Infection

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Induktion af graft-versus-host sygdom og<em&gt; In Vivo</em&gt; T Cell Monitoring Brug en MHC-matchet murin model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Trans-vivo forsinket hypersensibilitet Assay til antigenspecifik forordning

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

In vitro assay at evaluere virkningen af immunoregulerende Pathways på HIV-specifikke CD4 T-celle effektorfunktion

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

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An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

En<em&gt; Ex vivo</em&gt; Model af en oligodendrocyt rettet T-celle Angrib akut Brain Skiver

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

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Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

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Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Vurdering Retinal mikrogliaceller fagocytotisk funktion<em&gt; In vivo</em&gt; Brug en flowcytometri-baseret assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

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Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Generering<em&gt; De Novo</em&gt; Antigenspecifikke humane T-celle receptorer ved retroviral transduktion af Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

Detektion og berigelse af Rare Antigen-specifikke B-Celler til analyse af fænotype og funktion

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

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Et Protein Microarray analyse for serologisk bestemmelse af Antigen-specifikke antistof reaktioner følgende Clostridium difficile infektion

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...