Name: in situ mhc-tetramer staining and quantitative analysis to determine the location, abundance, and phenotype of antigen-specific cd8 t cells in tissues
Uploaded: 2017-09-22T15:00:00
Description: Watch this Scientific Journal Video about In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues at JoVE

Dit werk werd gesteund door Public Health Service verleent van de National Institutes of Health (T32 DA007097, R01AI096966, andUM1AI26617).

1. dag 1: verse weefsel segmenteren en primaire incubatie <ol><li>gebruik een scalpel aan verse weefsel in kleine (ongeveer 0.5 cm breed door 0.5 cm hoog) stukjes gesneden. Afzonderlijk lijm van elk weefsel naar een zuiger en insluit met 4% laag-smelt agarose 3-5 mL in PBS. Label de zuiger met de informatie van de weefsel met behulp van een sticker. Zet het in een gekoeld houder in een ijsemmer te stollen.</li>
	<li>Inschakelen de microtoom en de dikte van de secties naar 200 µm. installeren een scheermesje op de mic...

<ol>
	<li>Altman, J. 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=Phenotypic+analysis+of+antigen-specific+T+lymphocytes.">Phenotypic analysis of antigen-specific T lymphocytes.</a> Science. 274 (5284), 94-96 (1996).</li><li>Novak, E. J., Liu, A. W., Nepom, G. T., Kwok, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+Class+II+tetramers+identify+peptide-specific+human+CD4(+)+T+cells+proliferating+in+response+to+influenza+A+antigen.">MHC Class II tetramers identify peptide-specific human CD4(+) T cells proliferating in response to influenza A antigen.</a> J Clin Invest. 104, R63-R67 (1999).</li><li>Steinert, E. 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=Quantifying+memory+CD8+T+cells+reveals+regionalization+of+immunosurveillance.">Quantifying memory CD8 T cells reveals regionalization of immunosurveillance.</a> Cell. 161 (4), 737-749 (2015).</li><li>Skinner, P. J., Daniels, M. A., Schmidt, C. S., Jameson, S. C., Haase, A. T. <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:+In+situ+tetramer+staining+of+antigen-specific+T+cells+in+tissues.">Cutting edge: In situ tetramer staining of antigen-specific T cells in tissues.</a> J Immunol. 165 (2), 613-617 (2000).</li><li>Haanen, J. B., 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=In+situ+detection+of+virus-+and+tumor-specific+T-cell+immunity.">In situ detection of virus- and tumor-specific T-cell immunity.</a> Nat Med. 6 (9), 1056-1060 (2000).</li><li>Skinner, P. J., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+tetramer+staining+using+MHC+class+I+tetramers.">In situ tetramer staining using MHC class I tetramers.</a> J Immunol Methods. 268 (1), 29-34 (2002).</li><li>Skinner, P. J., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+staining+using+MHC+class+I+tetramers.">In situ staining using MHC class I tetramers.</a> Curr Protoc Immunol. 17, 4.1-4.9 (2004).</li><li>Li, 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=Use+of+HLA-DR*08032/E7+and+HLA-DR*0818/E7+tetramers+in+tracking+of+epitope-specific+CD4++T+cells+in+active+and+convalescent+tuberculosis+patients+compared+with+control+donors.">Use of HLA-DR*08032/E7 and HLA-DR*0818/E7 tetramers in tracking of epitope-specific CD4+ T cells in active and convalescent tuberculosis patients compared with control donors.</a> Immunobiology. 216, 947-960 (2011).</li><li>Bischof, F., 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=Analysis+of+autoreactive+CD4+T+cells+in+experimental+autoimmune+encephalomyelitis+after+primary+and+secondary+challenge+using+MHC+class+II+tetramers.">Analysis of autoreactive CD4 T cells in experimental autoimmune encephalomyelitis after primary and secondary challenge using MHC class II tetramers.</a> J Immunol. 172, 2878-2884 (2004).</li><li>Massilamany, 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=Direct+staining+with+major+histocompatibility+complex+class+II+dextramers+permits+detection+of+antigen-specific,+autoreactive+CD4+T+cells+in+situ.">Direct staining with major histocompatibility complex class II dextramers permits detection of antigen-specific, autoreactive CD4 T cells in situ.</a> PLoS ONE. 9, e87519 (2014).</li><li>Massilamany, 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=In+situ+detection+of+autoreactive+CD4+T+cells+in+brain+and+heart+using+major+histocompatibility+complex+class+II+dextramers.">In situ detection of autoreactive CD4 T cells in brain and heart using major histocompatibility complex class II dextramers.</a> J Vis Exp. (90), e51679 (2014).</li><li>Dileepan, T., Kim, H. O., Cleary, P. P., Skinner, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Peptide-MHC-II+Tetramer+Staining+of+Antigen-Specific+CD4++T+Cells+in+Tissues.">In Situ Peptide-MHC-II Tetramer Staining of Antigen-Specific CD4+ T Cells in Tissues.</a> PLoS ONE. 10 (6), e0128862 (2015).</li><li>Tjernlund, 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=In+situ+detection+of+Gag-specific+CD8++cells+in+the+GI+tract+of+SIV+infected+Rhesus+macaques.">In situ detection of Gag-specific CD8+ cells in the GI tract of SIV infected Rhesus macaques.</a> Retrovirology. 7, 7-12 (2010).</li><li>Connick, E., 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=CTL+fail+to+accumulate+at+sites+of+HIV-1+replication+in+lymphoid+tissue.">CTL fail to accumulate at sites of HIV-1 replication in lymphoid tissue.</a> J Immunol. 178, 6975-6983 (2007).</li><li>Hong, J. 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=Localized+Populations+of+CD8low/&#8722;+MHC+Class+I+Tetramer++SIV-Specific+T+Cells+in+Lymphoid+Follicles+and+Genital+Epithelium.">Localized Populations of CD8low/&#8722; MHC Class I Tetramer+ SIV-Specific T Cells in Lymphoid Follicles and Genital Epithelium.</a> PLoS ONE. 4 (1), e4131 (2009).</li><li>Sasikala-Appukuttan, A. K., 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=Location+and+dynamics+of+the+immunodominant+CD8+T+cell+response+to+SIV&#916;nef+immunization+and+SIVmac251+vaginal+challenge.">Location and dynamics of the immunodominant CD8 T cell response to SIV&#916;nef immunization and SIVmac251 vaginal challenge.</a> PLoS ONE. 8 (12), e81623 (2013).</li><li>Connick, E., 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=Compartmentalization+of+simian+immunodeficiency+virus+replication+within+secondary+lymphoid+tissues+of+rhesus+macaques+is+linked+to+disease+stage+and+inversely+related+to+localization+of+virus-specific+CTL.">Compartmentalization of simian immunodeficiency virus replication within secondary lymphoid tissues of rhesus macaques is linked to disease stage and inversely related to localization of virus-specific CTL.</a> J Immunol. 193, 5613-5625 (2014).</li><li>Li, Q., 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=Visualizing+antigen-specific+and+infected+cells+in+situ+predicts+outcomes+in+early+viral+infection.">Visualizing antigen-specific and infected cells in situ predicts outcomes in early viral infection.</a> Science. 323, 1726-1729 (2009).</li><li>Li, Q., Skinner, P. J., Duan, L., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+to+simultaneously+visualize+virus-specific+CD8++T+cells+and+virus-infected+cells+in+situ.">A technique to simultaneously visualize virus-specific CD8+ T cells and virus-infected cells in situ.</a> J Vis Exp. (30), e1561 (2009).</li><li>Li, 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=Simian+immunodeficiency+virus-producing+cells+in+follicles+are+partially+suppressed+by+CD8++cells+in+vivo.">Simian immunodeficiency virus-producing cells in follicles are partially suppressed by CD8+ cells in vivo.</a> J Virol. 90, 11168-11180 (2016).</li><li>Daniels, M. A., Jameson, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+for+CD8+in+T+cell+receptor+binding+and+activation+by+peptide/major+histocompatibility+complex+multimers.">Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers.</a> J Exp Med. 191, 335-346 (2000).</li><li>Lee, Y. J., Wang, H., Starrett, G. J., Phuong, V., Jameson, S. C., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+specific+distribution+of+iNKT+cells+impacts+their+cytokine+response.">Tissue specific distribution of iNKT cells impacts their cytokine response.</a> Immunity. 43 (3), 566-578 (2015).</li><li>Abdelaal, H. M., Kim, H. O., Wagstaff, R., Sawahata, R., Southern, P. J., Skinner, P. J. <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+Vibratome+and+Compresstome+sectioning+of+fresh+primate+lymphoid+and+genital+tissues+for+in+situ+MHC-tetramer+and+immunofluorescence+staining.">Comparison of Vibratome and Compresstome sectioning of fresh primate lymphoid and genital tissues for in situ MHC-tetramer and immunofluorescence staining.</a> Biol Proced Online. 17, (2015).</li><li>Wang, F., 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=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> J Mol Diagn. 14 (1), 22-29 (2012).</li></ol>

IST gecombineerd met IHC biedt een essentieel instrument voor het opsporen, karakteriseren, en kwantificeren van antigeen-specifieke CD8 T cellen in native omgevingen met de context van andere cellen en weefsel structuren. Hier, beschreven we gedetailleerde procedures voor het IST gecombineerd met IHC, gevolgd door kwantitatieve beeldanalyse, om te bepalen van de locatie, overvloed en fenotype van antigeen-specifieke CD8 T-cellen in de lymfeklieren van rhesus makaken. Soortgelijke kleuring kan worden toegepast op de mens...

T-cellen zijn cruciaal voor veel immunologische processen, waaronder opsporen en elimineren van virus-geïnfecteerde cellen voorkomen autoimmuniteit, assisteren bij B-cel en plasma-cel productie van antilichamen, opsporen en elimineren van kankercellen. De ontwikkeling van peptide/MHC klasse I tetrameer kleuring van antigeen-specifieke CD8 T cellen1 en de meer recente ontwikkeling van de MHC klasse II tetrameer kleuring van CD4 T cellen2 door stroom cytometry een revolutie teweeggebracht in ons vermogen te bestuderen en begrijpen de Immunobiologie van T-cellen. Terwijl uiterst nuttig voor het bepalen van de hoeveelheid en het fenotype van antigeen-specifieke T-cellen, stroom cytometry niet mogelijk voor de detectie van de ruimtelijke lokalisatie van antigeen-specifieke T-cellen naar andere cellen en structuren in de weefsels, en de huidige aggregatieniveau technieken om uit te pakken van de T-cellen die nodig zijn voor stroom cytometry hebben beperkte doeltreffendheid in niet-lymfoïde weefsels3.
Wij en anderen hebben ontwikkeld methoden met peptide-geladen MHC klasse I en klasse II tetrameer of multimer reagentia vlek van antigeen-specifieke CD8 en CD4 T-cellen in weefsels4,5,6,7, 8 , 9 , 10 , 11 , 12 , 13. deze IST methoden voor de bepaling van de locatie, overvloed en fenotype van antigeen-specifieke CD8 en CD4 T-cellen in weefsels verlenen en een middel om het detecteren van deze cellen ten opzichte van andere cellen en structuren in de weefsels. Onze fractie heeft uitgebreid gebruikt voor MHC-ik tetrameer vlekken om te bestuderen van humaan immunodeficiëntie virus (HIV)- en simian immunodeficiëntie virus (SIV) - specifieke CD8 T-cellen in de lymfoïde, genitale en rectale weefsels een inzicht van HIV en SIV immunopathogenese en te identificeren correlaten van succesvolle vaccinatie strategieën14,15,16,17. Daarnaast ontwikkeld we ook een techniek die IST met in situ hybridisatie (ISH combineert) te lokaliseren en te kwantificeren van virus-specifieke CD8 T cellen en virus-geïnfecteerde cellen in weefsels en om te bepalen van de in vivo effector-tot-streefpad niveaus 18 , 19.
Hier beschrijven we een protocol met behulp van peptide-geladen MHC-ik tetramers om vlek van antigeen-specifieke CD8 T-cellen in de weefselsecties van vers, tot counterstain weefsels met behulp van IHC en te kwantificeren van cellen met specifieke fenotypen in specifieke weefsel compartimenten. Deze procedures zijn dezelfde als in onze recente publicatie werden gebruikt door Li et al., waarin wij de locatie, overvloed en fenotype van SIV-specifieke T-cellen in de lymfoïde weefsel tijdens chronische SIV infectie in Makaken20 vastbesloten.
Voor deze procedure, verse weefsels zijn gesegmenteerd en 's nachts geïncubeerd met peptide-geladen MHC-ik tetramers geconjugeerd met fluoresceïne kaliumthiocyanaat moleculen (FITC). Ze worden dan opgelost met paraformaldehyde. Na de vaststelling van het weefsel, wordt het signaal van de MHC tetramers versterkt met behulp van konijn anti-FITC antilichamen en geïncubeerd met fluorescently tagged anti-konijn IgG antilichamen, die het signaal van de afhankelijke tetramers verder te versterken. IHC wordt gebruikt in combinatie met IST te karakteriseren van antigeen-specifieke T-cellen en de omringende cellen. Antilichamen die herkennen epitopes op het oppervlak van cellen of in de extracellulaire ruimte zijn opgenomen in de primaire incubatie met de tetramers. Antilichamen die intracellulaire epitopes herkent vereisen permeatie van de celwand voorafgaand aan kleuring. De gekleurde weefselsecties zijn beeld met behulp van een confocal microscoop en geanalyseerd met behulp van de confocal software. Gelabelde cellen worden gekwantificeerd aan de hand van de confocal microscopie software of ImageJ. De beschreven protocol kan worden gebruikt om vlek in wezen een antigeen-specifieke CD8 T cel in elk weefsel voor welke MHC-ik tetramers zijn beschikbaar.

<table align="center" border="1" cellpadding="0" cellspacing="0" style="width:906px;" width="906">
	<tbody>
		<tr>
			<td>MHC-I monomer</td>
			<td>NIH tetramer core facility</td>
			<td></td>
			<td>Materials for MHC-tetramer preparation</td>
		</tr>
		<tr>
			<td>ExtrAvidin-FITC</td>
			<td>Sigma-Aldrich</td>
			<td>E2716</td>
			<td>Materials for MHC-tetramer preparation</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson Immunoresearch</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melt agarose</td>
			<td>Promega</td>
			<td>V3121</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>SLBL6391V</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T-6878</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>J.T.Baker</td>
			<td>4204-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol/gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>SLBH2672V</td>
			<td></td>
		</tr>
		<tr>
			<td>n-propyl gallate</td>
			<td>Sigma-Aldrich</td>
			<td>P3130</td>
			<td></td>
		</tr>
		<tr>
			<td>rat-a-h-CD8 (1:500)</td>
			<td>Acris</td>
			<td>0714</td>
			<td>Antibody unstable, use single use frozen aliquot</td>
		</tr>
		<tr>
			<td>m-a-h-CD20 (1:500)</td>
			<td>NOVOCASTRA</td>
			<td>6026819</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-h-Ki67 (1:500)</td>
			<td>Vector</td>
			<td>6022201</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-a-m-A488 (1:2000)</td>
			<td>Jackson Immunoresearch</td>
			<td>124083</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-a-rb-Cy3 (1:5000)</td>
			<td>Jackson Immunoresearch</td>
			<td>106232</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-a-rat-Cy5 (1:5000)</td>
			<td>Jackson Immunoresearch</td>
			<td>118088</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-a-h-IgM-Dylight649 (1:5000)</td>
			<td>Jackson Immunoresearch</td>
			<td>86579</td>
			<td></td>
		</tr>
		<tr>
			<td>Compresstome: VF-300 Microtome</td>
			<td>Precisionary Instruments, LLC</td>
			<td>1079</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick Set Instant Adhesive</td>
			<td>Loctite</td>
			<td>46551</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well flat bottomed tissue culture plates</td>
			<td>Falcon</td>
			<td>353226</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Globe scienfitic Inc.</td>
			<td>#1321</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor&#160; blade</td>
			<td>Ted Pella, Inc</td>
			<td>121-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather Disposable Scalpel</td>
			<td>FEATHER SAFETY RAZOR CO. LTD.</td>
			<td>No. 21</td>
			<td></td>
		</tr>
		<tr>
			<td>Round paintbrush #2</td>
			<td>PRINCETON ART &#38; BRUSH CO.</td>
			<td>4350R</td>
			<td>Can trim as needed with razor</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>FV10-ASW_Viewer4.0</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ mhc-tetramer staining and quantitative analysis to determine the location, abundance, and phenotype of antigen-specific cd8 t cells in tissues

T-cellen zijn cruciaal voor veel immunologische processen, waaronder opsporen en elimineren van virus-geïnfecteerde cellen voorkomen autoimmuniteit, assisteren bij B-cel en plasma-cel productie van antilichamen, opsporen en elimineren van kankercellen. De ontwikkeling van MHC-tetrameer kleuring van antigeen-specifieke T-cellen geanalyseerd door stroom cytometry heeft een revolutie teweeggebracht in ons vermogen te bestuderen en begrijpen van de Immunobiologie van T-cellen. Terwijl uiterst nuttig voor het bepalen van de hoeveelheid en het fenotype van antigeen-specifieke T-cellen, kan niet stroom cytometry bepalen wat de ruimtelijke lokalisatie van antigeen-specifieke T-cellen naar andere cellen en structuren in de weefsels, en huidige aggregatieniveau technieken om uit te pakken van de T die nodig zijn cellen voor stroom cytometry hebben beperkte doeltreffendheid in niet-lymfoïde weefsels. In situ MHC-tetrameer kleuring (IST) is een techniek om te visualiseren van T-cellen die specifiek voor antigeen van belang in de weefsels zijn. In combinatie met immunohistochemistry (IHC), kunt IST bepalen de overvloed, locatie en fenotype van antigeen-specifieke CD8 en CD4 T-cellen in weefsels. Hier beschrijven we een protocol om de vlek en inventariseren van antigeen-specifieke CD8 T-cellen, met specifieke fenotypen gelegen binnen specifieke weefsel compartimenten. Deze procedures zijn dezelfde die we in onze recente publicatie door Li et al. gebruikt, getiteld "Simian Immunodeficiëntie Virus-producerende cellen in de follikels gedeeltelijk onderdrukt door CD8+ cellen In Vivo." De beschreven methoden zijn breed toepasbare omdat ze kunnen worden gebruikt voor het lokaliseren, fenotype, en in wezen een antigeen-specifieke CD8 T-cel waarvoor MHC tetramers beschikbaar zijn, in elk weefsel te kwantificeren.

Figuur 1 toont hoe verzamelt confocal beelden met behulp van een confocal microscoop. Figuur 2 toont kwantitatieve beeldanalyse ImageJ gebruiken. Figuren 3 en 4 tonen vertegenwoordiger beelden van lymfeklier weefsels van een SIV besmet resusaap gekleurd met MHC tetramers, CD8 antilichamen en CD20 antilichamen, en dienen om aan te tonen van de specificiteit van de MHC-tetrameer kleuring. <strong c...

Watch this Scientific Journal Video about In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues at JoVE

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

Hier beschrijven we een methode die in situ MHC-tetrameer kleuring combineert met immunohistochemistry lokalisatie, fenotype, en hoeveelheid van antigeen-specifieke T-cellen in weefsels te bepalen. Dit protocol wordt gebruikt om te bepalen van de ruimtelijke en fenotypische kenmerken van antigeen-specifieke CD8 T-cellen ten opzichte van andere celtype en structuren in de weefsels.

In Situ MHC-tetrameer kleuring en kwantitatieve analyse om te bepalen van de locatie, overvloed en fenotype van CD8 Antigen-specifieke T-cellen in weefsels

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

The overall goal of this procedure is to determine the location, abundance and phenotype of antigen-specific CD8 positive T cells within tissues of interest. This method helps answer quick questions about CD8 T cells by providing a means to locate, quantify and phenotype virtually any antigen specific CD8 T cell and any tissue. The main advantage of this technique is that it allows determination of the spatial distribution of antigen specific CD8 positive T cells relative to other cells and structures within tissues of interest.Demonstrating the procedure with Shengbin Li will be Gwantwa Mwakalundwa. On the day one of the procedure, use a scalpel to cut the fresh tissue into approximately 0.5 x 0.5 centimeter pieces. Here, rhesus macaque spleen tissue is shown.Separately glue each tissue fragment into the middle of the top of a plunger and embed the samples in three to five milliliters of warm 4%low melting agarose in PBS. Label the plungers with the relevant tissue information. Then, place the samples in a chilled holder in an ice bucket.When the agar has solidified, turn on the microtome and set the thickness setting to 200 micrometers. After installing a new razor blade, insert the plungers into the microtome bath. Fill the microtome bath with 100 to 200 milliliters of freshly prepared PBS-H and add PBS-H ice cubes to maintain the temperature between zero to two degrees celsius.After obtaining 200 micrometer sections, use a paintbrush to remove the agar surrounding each section and transfer the samples into individual tissue chambers within individual wells of a 24 well tissue culture plate containing one milliliter of chilled PBS-H. When all of the samples have been acquired, incubate the tissue sections overnight in one milliliter of fit conjugated peptide loaded MHC class one tetramers, diluted in PBS-H supplemented with 2%normal goat serum at four degree celsius with rocking. The following morning, transfer each tissue chamber into individual wells of a new 24 well plate containing one milliliter of fresh chilled PBS-H for 20 minutes.After the second wash, fix the sections in one milliliter of fresh 4%paraformaldehyde for two hours at room temperature. Followed by two five minute washes in cold PBS-H. Transfer the sections into a new 24 well plate containing 0.01 molar urea and microwave the sections three times for about 10 seconds each time until the urea boils.To permeabilize and block the samples, incubate the sections in blocking solution containing PBS-H supplemented with a 0.3%triton x100 detergent and 2%normal goat serum on a rocker for one hour, at four degree celsius. At the end of the incubation, transfer the tissue chambers into individual wells of a new 24 well plate containing one milliliter of solution containing rabbit anti-fitc antibody, mouse antihuman CD antibody and rat antihuman CD3 antibody. Incubate the samples overnight at four degrees celsius with rocking.On day three, wash the sections three times in fresh PBS-H at four degree celsius for at least 20 minutes per wash and perform a final incubation with the appropriate fluorescence conjugated secondary antibodies for up to three days, protected from light. At the end of the tertiary antibody incubation, rinse the samples with three 20 minute washes in fresh PBS-H and use a paint brush to transfer the samples from each tissue chamber onto a glass microscope slide. Coat each section with mounting medium supplemented with a fluorophore preservative and cover the samples with a cover slip.Then, store the slides in a light protected slide container, at 20 degrees celsius. On the day of imaging, place the first slide onto the stage of a confocal microscope and select the appropriate lasers and filters for each fluorophore used to label the tissue sample. Using the 20x objective, sequentially obtain a z series at three micron intervals in each of the fluorophore channels, in multiple 800 x 800 pixel fields.Then, construct a montage of the collected fields and save each montage image with a name based on the corresponding tissue sample. In these representative images, a section stained with MHC class one tetramers loaded with an SIV peptide is compared to a section from the same tissue, stained with MHC class one tetramers loaded with an irrelevant peptide, demonstrating the specificity of the tetramer staining. In these images, the virus specific T cells stained with MHC class one tetramer loaded with SIV peptide, co-stains for CD8, a T cell subset marker but not CD20, a cell surface protein expressed on B cells.This montage was created for multiple confocal z series fields, that were used for the quantification of tetramer stained cells with specific phenotypes and different anatomical compartments on the lymph node. This magnified image highlights an area of the montage lymph node that contains a B cell follicle delineated by IGM staining. MHC class one tetramer stained cells, some of which co-expressed the T cell activation and proliferation marker, KI67 can be detected inside and outside of B cell follicle.This staining combination allows the determination of the activation and proliferation statuses of SIV specific CD8 positive T cells, as well as their localisation within secondary lymphoid tissues. Once mastered, this technique can be completed in approximately five days if it is performed properly. While attempting this procedure, it's important to remember that fresh tissue is critical for successful staining.Process the samples within 24 hours or less after harvesting, keeping them chilled until fixation. After watching this video, you should have a good understanding of how to perform a tetramer staining and immunohistochemistry to determine the location, abundance and phenotype of antigen specific CD8 positive T cells in tissues of interest. Don't forget that working with infectious agents can be extremely hazardous and that precautions to prevent exposure such as, wearing the appropriate personal protection equipment and limiting the use of sharps should always be taken while performing this procedure.

Research

JoVE Journal

Immunology and Infection

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

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

Retrovirale Transductie van T-cel-receptoren in Mouse T-cellen

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 ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Killer Artificial antigeen presenterende cellen (KaAPC) voor Efficient<em&gt; In Vitro</em&gt; Uitputting van Human Antigeen-specifieke T-cellen

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Whole-dierlijke Imaging en Flowcytometrische technieken voor analyse van antigeen-specifieke CD8 + T cel reacties na Nanoparticle Vaccinatie

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

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrofielen Isolatie en analyse van hun rol in de Lymphoma Cell Gevoeligheid voor therapeutische middelen te bepalen

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Ontwikkeling van stamcel-afgeleide Antigeen-specifieke regulatoire T-cellen tegen auto-immuniteit

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

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

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

Analyse van Simian Immunodeficiency Virus-specifieke CD8<sup&gt; +</sup&gt; T-cellen in resusapen door peptide-MHC-I tetrameer kleuring

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

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

Detectie en Verrijking van zeldzame antigeen-specifieke B-cellen voor analyse van fenotype en functie

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 ...

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

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

In Vivo Assay voor detectie van antigeen-specifieke T-cel cytolytische functie met behulp van een vaccinatie-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 ...

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

Metabole Mapping: Kwantitatieve enzym cytochemie en histochemie te bepalen van de activiteit van Dehydrogenases in cellen en weefsels

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Zeer efficiënte generatie van antigeen-specifieke cytotoxische T-cellen voor de functionele test in een auto-immune diabetes model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...