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

Dette arbejde blev støttet af Public Health Service tilskud fra National Institutes of Health (T32 DA007097, R01AI096966, andUM1AI26617).

1. dag 1: frisk væv skæring og primære inkubation <ol><li>Brug en skalpel til at skære frisk væv i små (ca 0,5 cm bred ved 0,5 cm høj) stykker. Separat lim hver væv til et stempel og integrere dem med 3-5 mL 4% lav-Smelt Agarosen i PBS. Label stemplet med væv oplysninger ved hjælp af en mærkat. Sætte det i en kølet indehaveren i en isspand størkne.</li>
	<li>Tænder på mikrotomen og sæt tykkelsen af sektioner til 200 µm. installere et barberblad på mikrotomen og Indsæt stemplet monteret med væv i m...

<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. 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IST kombineret med IHC giver et vigtigt redskab for påvisning, kendetegner og kvantificere antigen-specifikke CD8 T celler i native miljøer med konteksten af andre celler og væv strukturer. Her, beskrevet vi nærmere procedurer for IST kombineret med IHC, efterfulgt af kvantitative billedanalyse, til at bestemme placering, overflod og fænotype af antigen-specifikke CD8 T-celler i lymfeknuder fra rhesus makakaber. Lignende farvning kan blive anvendt til menneskelige, mus, eller andre arter væv for hvilke MHC-jeg tetr...

T celler er vigtige for mange immunologiske processer, herunder opdage og fjerne virus-inficerede celler, forhindrer autoimmunitet, bistå i B-celle og plasma-celle produktion af antistoffer, og opdage og fjerne kræftceller. Udviklingen af peptid/MHC klasse I tetramer farvning af antigen-specifikke CD8 T celler1 og den nyere udvikling af MHC klasse II tetramer farvning af CD4 T celler2 ved flowcytometri revolutioneret vores evne til at studere og forstå de Immunobiology af T-celler. Yderst nyttige til at bestemme mængden og fænotype af antigen-specifikke T-celler, tillader flowcytometri ikke til påvisning af den geografiske lokalisering af antigen-specifikke T-celler til andre celler og strukturer i væv, og nuværende opdeling teknikker til at udvinde T celler behov for flowcytometri har begrænset effektiviteten i ikke-lymfoide væv3.
Vi og andre har udviklet metoder bruger peptid-læsset MHC klasse I og klasse II tetramer eller multimer reagenser til pletten antigen-specifikke CD8 og CD4 T celler i væv4,5,6,7, 8 , 9 , 10 , 11 , 12 , 13. disse IST metoder give til bestemmelse af placering, overflod og fænotype af antigen-specifikke CD8 og CD4 T celler i væv og give et middel til at opdage disse celler i forhold til andre celler og strukturer i væv. Vores gruppe har flittigt brugt MHC-jeg tetramer farvning for at studere human immundefekt virus (HIV)- og simian immundefektvirus (SIV) - specifikke CD8 T celler i lymfoide, kønsorganer og rektal væv til at opnå en forståelse af HIV og SIV immunpatogenese og at identificere korrelerer vellykkede vaccination strategier14,15,16,17. Derudover udviklet vi også en teknik, der kombinerer IST med i situ hybridisering (ISH) at lokalisere og kvantificere virus-specifikke CD8 T celler og virus-inficerede celler i væv og til at bestemme in vivo effektor-til-målet-niveauer 18 , 19.
Her, vi beskriver en protokol bruger peptid-læsset MHC-jeg tetramers til at plette antigen-specifikke CD8 T celler i frisk væv sektioner, til kontrastfarve væv ved hjælp af IHC og kvantificere celler med specifikke fænotyper i bestemte væv rum. Disse procedurer er de samme som blev brugt i vores seneste publikation af Li et al., hvor vi bestemt placering, overflod og fænotype af SIV-specifikke T-celler i lymfoide væv under kronisk SIV infektion i makakaber20.
For denne procedure, frisk væv i snit og inkuberes natten over med peptid-læsset MHC-jeg tetramers konjugeret til fluorescein kaliumthiocyanat molekyler (FITC). De er så fast i PARAFORMALDEHYD. Efter fastsættelsen af væv, forstærkes signalet fra MHC-tetramers ved hjælp af kanin anti-FITC antistoffer og inkuberes med fluorescently mærket anti-kanin IgG antistoffer, som yderligere forstærker signalet fra de bundne tetramers. IHC bruges i forbindelse med IST til at karakterisere antigen-specifikke T-celler og omkringliggende celler. Antistoffer, der genkender epitoper på overfladen af celler eller i det ekstracellulære rum er inkluderet i den primære inkubering med en tetramers. Antistoffer, der genkender intracellulære epitoper kræver gennemtrængning af cellevæggen før farvning. Afsnittene farves væv er afbildet ved hjælp af en Konfokal mikroskop og analyseret ved hjælp af Konfokal software. Mærket celler er kvantificeret konfokalmikroskopi software eller ImageJ. Den beskrevne protokol kan bruges til at plette hovedsagelig alle antigen-specifikke CD8 T celler i alle væv for hvilke MHC-jeg tetramers er tilgængelige.

<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 celler er vigtige for mange immunologiske processer, herunder opdage og fjerne virus-inficerede celler, forhindrer autoimmunitet, bistå i B-celle og plasma-celle produktion af antistoffer, og opdage og fjerne kræftceller. Udviklingen af MHC-tetramer farvning af antigen-specifikke T-celler analyseret ved flowcytometri har revolutioneret vores evne til at studere og forstå immunobiology af T-celler. Yderst nyttige til at bestemme mængden og fænotype af antigen-specifikke T-celler, kan ikke flowcytometri bestemme den geografiske lokalisering af antigen-specifikke T-celler til andre celler og strukturer i væv og nuværende opdeling teknikker for at udtrække T behov celler for flowcytometri har begrænset effektiviteten i ikke-lymfoide væv. In situ MHC-tetramer farvning (IST) er en teknik til at visualisere T-celler, der er specifikke for antigener af interesse i væv. I kombination med Immunhistokemi (IHC), kan IST bestemme overflod, placering og fænotype af antigen-specifikke CD8 og CD4 T celler i væv. Her, beskriver vi en protokol for at pletten og optælle antigen-specifikke CD8 T-celler med specifikke fænotyper ligger inden for specifikke væv rum. Disse procedurer er de samme som vi brugte i vores seneste publikation af Li et al., med titlen "Simian immundefektvirus-producerende celler i follikler er delvist undertrykt af CD8 celler,+ In Vivo." De beskrevne metoder er stort set gældende, fordi de kan bruges til at lokalisere, fænotype, og kvantificere det væsentlige alle antigen-specifikke CD8 T celler som MHC tetramers der findes i alle væv.

Figur 1 viser hvordan man skal indsamle Konfokal billeder ved hjælp af en Konfokal mikroskop. Figur 2 viser kvantitative billedanalyse ved hjælp af ImageJ. Figur 3 og 4 viser repræsentant billeder af lymfeknude væv fra en SIV inficerede rhesus makak farves med MHC tetramers, CD8 antistoffer og CD20 antistoffer, og tjene til at demonstrere specificiteten af MHC-tetramer farvning. <strong class...

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

Her, beskriver vi en metode, der kombinerer i situ MHC-tetramer farvning med Immunhistokemi at bestemme lokalisering, fænotype og mængden af antigen-specifikke T-celler i væv. Denne protokol bruges til at bestemme de rumlige og fænotypiske egenskaber af antigen-specifikke CD8 T celler i forhold til andre celletype og strukturer i væv.

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 ...

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

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