Bu çalışma Cure Vakfı ve NIH / NCI 9R01 CA152045 ve P50-CA116201 için Susan G. Komen Keith L. Knutson için hibe tarafından desteklenmiştir.

<ol>
	<li>Zhang, N., Bevan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8(+)+T+cells:+foot+soldiers+of+the+immune+system.">CD8(+) T cells: foot soldiers of the immune system.</a> Immunity. 35, 161-168 (2011).</li><li>Garboczi, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+complex+between+human+T-cell+receptor,+viral+peptide+and+HLA-A2.">Structure of the complex between human T-cell receptor, viral peptide and HLA-A2.</a> Nature. 384, 134-141 (1996).</li><li>Knutson, K. L., dela Rosa, C., Disis, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+analysis+of+T-cell+immunity.">Laboratory analysis of T-cell immunity.</a> Front Biosci. 11, 1932-1944 (2006).</li><li>Andersen, M. H., Schrama, D., Thor Straten, P., Becker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cytotoxic T cells. J. Invest. Dermatol. 126, 32-41 (2006).</li><li>Weren, A., Bonnekoh, B., Schraven, B., Gollnick, H., Ambach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+flow+cytometric+assay+focusing+on+perforin+release+mechanisms+of+cytotoxic+T+lymphocytes.">A novel flow cytometric assay focusing on perforin release mechanisms of cytotoxic T lymphocytes.</a> J. Immunol. Methods. 289, 17-26 (2004).</li><li>Aktas, E., Kucuksezer, U. C., Bilgic, S., Erten, G., Deniz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+CD107a+expression+and+cytotoxic+activity.">Relationship between CD107a expression and cytotoxic activity.</a> Cell Immunol. 254, 149-154 (2009).</li><li>Knutson, K. L., Schiffman, K., Disis, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+with+a+HER-2/neu+helper+peptide+vaccine+generates+HER-+2/neu+CD8+T-cell+immunity+in+cancer+patients.">Immunization with a HER-2/neu helper peptide vaccine generates HER- 2/neu CD8 T-cell immunity in cancer patients.</a> J. Clin. Invest. 107, 477-484 (2001).</li><li>Asiedu, M. K., Ingle, J. N., Behrens, M. D., Radisky, D. C., Knutson, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta/TNF(alpha)-mediated+epithelial-mesenchymal+transition+generates+breast+cancer+stem+cells+with+a+claudin-low+phenotype.">TGFbeta/TNF(alpha)-mediated epithelial-mesenchymal transition generates breast cancer stem cells with a claudin-low phenotype.</a> Cancer Res. 71, 4707-4719 (2011).</li><li>Strijbosch, L. W., Buurman, W. A., Does, R. J., Zinken, P. H., Groenewegen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+dilution+assays.+Experimental+design+and+statistical+analysis.">Limiting dilution assays. Experimental design and statistical analysis.</a> J. Immunol. Methods. 97, 133-140 (1987).</li><li>Gallimore, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+exhaustion+of+lymphocytic+choriomeningitis+virus-specific+cytotoxic+T+lymphocytes+visualized+using+soluble+tetrameric+major+histocompatibility+complex+class+I-peptide+complexes.">Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes.</a> J. Exp. Med. 187, 1383-1393 (1998).</li></ol>

Üretim ve bu video-makale Ücretsiz Erişim Roche Diagnostics tarafından desteklenmektedir.

CD8 T hücreleri doğrudan enfekte veya kötü huylu hücreleri 4 öldürmek için güçlü olmak, adaptif bağışıklık yanıtı için önemlidir. Antijen-spesifik CD8 T hücreleri (örneğin ELISPOT, tetramer, flow sitometri) sayısını ölçmek için çeşitli yollar olsa da, çoğu kez bu hücrelerin 3 işlevsel sitotoksik olup olmadığını bilmek yararlıdır. Sitotoksik fonksiyonu değerlendirmek için en çok tanınan testi altın standart test 4 olarak kabul edilir MKK vardır. SRA önemli sınırlamalar radyoaktivite (1) şartı, duyarlılık (2) eksikliği, mekanik bir bilgi (3) eksikliği, hücrelerin büyük miktarlarda (4) gereksinimi, ve (5) zahmetli bir dizi vardır. Böylece, son on yılda, bu sınırlamaları en aza indirmek için yeni stratejiler benimseyerek ilgi olmuştur. Yeni yaklaşımlar içeren bu ölçen kaspaz sürüm 4, BLT esteraz activity5 ve CD107 6 yüzey ifadesi. Mevcut el yazması önerirRoche XCELLigence sistemi üzerinde gerçekleştirilen empedans tabanlı tahlil, aynı zamanda CRA alternatif olarak yararlı olabilir. Empedans dayalı bir yaklaşım uyum yeteneği yüzeylere sıkıca uymayan T hücreleri, bu nedenle ölçüm ile T hücrelerinin doğrudan müdahale herhangi bir endişe azalan empedansı üzerinde çok etkisi olması görünmüyor gözlem dayanmaktadır. Empedans analiz sisteminin kullanımı için gerekli emek önemli ölçüde daha az öncelikli radyoaktivite kullanımı için düzenleme ile ilgilidir ortadan kaldırılmasına bağlı olduğu tahmin edilmektedir. Empedans-tabanlı sistemin tek önemli dezavantajı $ 50.000 $ 60.000 bir harcama XCELLigence biriminin satın almak için gerekli olmasıdır. Bununla birlikte, bir birim XCELLigence radyasyon sayacı ihtiyacını ortadan kaldırır ve T hücre sitotoksisite 8 ek olarak, ilaç ve göç hücresel cevabı değerlendirmek için yararlı olan, çok yönlüdür. Bu gözlemlertümör hücre empedans doza bağımlı azalma, hem basit bir ikincil dereceden fonksiyon aşağıdaki oldukça dik bir eğim ile, Şekil 2 &#39;de gösterilen deney 9 sitotoksik hücrelerin sıklığı tahmin etmek için seyreltme tahlilleri (LDAS) sınırlayıcı kullanılan düşündürmektedir . Empedans tabanlı yaklaşım bir avantajı, gerekli T hücreleri ve tümör hücre sayısı LDA potansiyel olarak daha uygun hale CRA daha az olmasıdır. Örneğin, CRA, bir 01:40 oranında, oyuk başına 1 x 10 5 tümör hücreleri, oyuk başına 4 x 10 6 toplam T hücre gerektiren kaplama vardır. Bununla birlikte, empedans bazlı tahlilde, oyuk başına sadece 1.5 x 10 4 tümör hücreleri, oyuk başına sadece 6 x 10 5 toplam T hücreleri tüketir, ihtiyaç vardır. Bu nedenle, empedans bazlı tahlil% 85 daha az T hücreleri ve tümör hücreleri kullanır. Bu azalma, empedans tabanlı sistemi, insan klinik denemelerinde immünolojik izleme adapte edilebileceğini göstermektedir nerede tkan O sınırlı olabilir çizer. MKK tek bir zaman noktasında sınırlı iken Ayrıca, empedans bazlı analiz kullanıcı minimum ek emek ve hücreleri ile aynı deneyi birden fazla parçalama zaman noktalarında karşılaştırmak için izin, sürekli gerçek zamanlı veri toplar. Frekans hesaplamalar potansiyel olarak toplam 10 antijene spesifik CD8 T hücrelerinin fonksiyonel sitotoksik T hücrelerinin oranını belirlemek için örneğin tetramer analizleri fonksiyonu ölçmek olmayan diğer stratejiler ile kombine edilebilir. Empedans tabanlı yaklaşımın en çekici özelliklerinden biri çoğu diğer testlerin aksine, sitotoksisite potansiyel olarak daha kolay inhibitörleri veya antikorların kullanımı ile hücresel etkileşimleri çalışmak için yapım, gerçek zamanlı olarak izlenebilir olmasıdır. Başka bir yöntem, etiketleme karşı tümör hücreleri için uygun olmasıdır. Non-tümör hücreleri öldüren etiketlenmiş ve kalır çünkü Son olarak, steril, daha fazla hasat ve T-hücre-dirençli hücreler çalışma possib olduğule. Her iki sistem spesifik T hücreleri yanıt olarak tümör öldürme veri sağlamak Özetle,, empedans bazlı analiz MKK daha duyarlı ve daha esnek bir platform sağlar.

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Ficoll-Paque Plus </td>
 <td>GE Healthcare </td>
 <td>17-1440-03 </td>
 <td>Used to isolate PBMCs from blood </td>
 </tr>
 <tr>
 <td>RPMI 1640 </td>
 <td>Cellgro </td>
 <td>10-040-CV </td>
 <td>Cell culture media </td>
 </tr>
 <tr>
 <td>FBS </td>
 <td>Denville Scientific </td>
 <td>FB5001-H </td>
 <td>Cell culture media </td>
 </tr>
 <tr>
 <td>Human IL-2 </td>
 <td>eBioscience </td>
 <td>14-8029-81 </td>
 <td>For stimulating growth of T cells </td>
 </tr>
 <tr>
 <td>Human Interferon gamma </td>
 <td>Cell Sciences </td>
 <td>CR2026 </td>
 <td>For stimulating MHC expression in tumor cells </td>
 </tr>
 <tr>
 <td>HER-2/neu p369-377 peptide </td>
 <td>Mayo Clinic peptide synthesis core </td>
 <td>amino acid sequence KIFGSLAFL </td>
 <td>Binds to HLA-A2 </td>
 </tr>
 <tr>
 <td>Influenza p58-66 peptide </td>
 <td>Mayo Clinic peptide synthesis core </td>
 <td>amino acid sequence GILGFVFTL </td>
 <td>Binds to HLA-A2 </td>
 </tr>
 <tr>
 <td>Human CD3/CD28 Dynabeads </td>
 <td>Invitrogen </td>
 <td>111-61D </td>
 <td>For non-specific T cell activation and expansion </td>
 </tr>
 <tr>
 <td>Chromium-51 </td>
 <td>MP Biomedicals </td>
 <td>62015 </td>
 <td>CRA labeling reagent </td>
 </tr>
 <tr>
 <td>E-plate </td>
 <td>Roche Applied Science </td>
 <td>05469830001 </td>
 <td>For xCelligence impedance unit </td>
 </tr>
 <tr>
 <td>Luma plate 96 </td>
 <td>Perkin Elmer </td>
 <td>6005630 </td>
 <td>For measuring radioactivity in the Top Count NXT. </td>
 </tr>
 <tr>
 <td>xCelligence RTCA DP Station </td>
 <td>Roche Applied Science </td>
 <td>RTCA DP </td>
 <td>Impedance unit </td>
 </tr>
 <tr>
 <td>Top Count NXT </td>
 <td>Perkin Elmer </td>
 <td>C990601 </td>
 <td>Scintillation &amp; Luminescence Counter </td>
 </tr>
 <tr>
 <td>Fas Ligand antibody</td>
 <td>BD Biosciences</td>
 <td>556372</td>
 <td>For blocking Fas-Fas ligand interactions</td>
 </tr>
 <tr>
 <td>HLA-A2 BB7.2 antibody</td>
 <td>BD Biosciences</td>
 <td>551230</td>
 <td>For blocking HLA class I</td>
 </tr>
 <tr>
 <td>Mouse IgG2b, &kappa;</td>
 <td>BD Biosciences</td>
 <td>555740</td>
 <td>Isotype control for HLA class I</td>
 </tr>
 <tr>
 <td>SKBR3 tumor line</td>
 <td>ATCC</td>
 <td>HTB-30</td>
 <td>Adenocarcinoma</td>
 </tr>
 <tr>
 <td>MCF7 tumor line</td>
 <td>ATCC</td>
 <td>HTB-22</td>
 <td>Adenocarcinoma</td>
 </tr>
 <tr>
 <td>HCC1419 tumor line</td>
 <td>ATCC</td>
 <td>CRL-2326</td>
 <td>Primary ductal carcinoma</td>
 </tr>
 <tr>
 <td>BT20 tumor line</td>
 <td>ATCC</td>
 <td>HTB-19</td>
 <td>Carcinoma</td>
 </tr>
 </tbody>
</table>

determining optimal cytotoxic activity of human her2neu specific cd8 t cells by comparing the cr51 release assay to the xcelligence system

Sitotoksik CD8 T hücreleri enfekte ya da kötü huylu konak hücreleri 1 ölümünü indükleme yeteneğine sahip olan T-hücrelerinin bir alt grup oluştururlar. Bu hücreler, özel bir reseptör I 2 molekülleri HLA sınıf bağlı belirli bir antijenik peptid tanıyabilir, T hücresi alıcısı (TCR), adlı ifade eder. Gibi hedef hücre ölümü ile sonuçlanan granzymes ve perforin olarak litik moleküllerin üretimi ile HLA sınıf I molekülü sonuçları ile enfekte olmuş hücreleri veya tümör hücreleri kazanılmıştır. Bu tür ELISPOT veya akış sitometri gibi tahliller kullanılarak antijen spesifik CD8 T hücrelerinin oranlarını belirlemek için yararlı olsa da, sitotoksisite deneyleri 3, CD8 T hücre yanıtları kuvvetini tespit etmek için de yararlıdır. Sitotoksik fonksiyonu değerlendirmek için en çok tanınan testi standart bir test 4 olarak kabul edilir Krom Sürüm Testi (MKK) &#39;dir. CRA gama radyasyon hücrelerin maruz kalma, l dahil olmak üzere birçok sınırlamalar vardırtekrarlanabilirlik ve hücrelerin çok sayıda için bir gereklilik ack. Geçtiğimiz on yıl içinde, bu sınırlamaları aşmak için yeni stratejiler benimseyerek ilgi olmuştur. Yeni yaklaşımlar içeren bu ölçen kaspaz sürüm 4, BLT esteraz aktivite 5 ve CD107 6 yüzey ifadesi. Roche XCELLigence sistemi kullanılarak empedans bazlı analiz, MKK alternatif olarak potansiyel için yazıda incelenmiştir. Yapışık tümör hücreleri elektrot plakaları bağlanmasına zaman empedansı ya da bir elektrik akımına karşı oluşur. Tümör hücreleri öldürme aşağıdaki ayırmak ve elektriksel direnç XCELLigence sistemi tarafından ölçülebileceği azalır. Hücre-aracılı öldürme değerlendirmek için empedans dayalı bir yaklaşım uyum yeteneği T hücreleri en yüzeylere sıkıca uymayan ve böylece T hücrelerinin doğrudan müdahale herhangi bir endişe azalan empedansı üzerinde çok etkisi olması görünmüyor gözlem dayanmaktadır ileölçme. Sonuçlar empedans tabanlı tahlil, standart MKK göre artan bir hassasiyet ile antijene özgü sitotoksik CD8 T hücrelerinin seviyelerindeki değişiklikleri tespit edebilir olduğunu göstermektedir. Bu sonuçlara dayanarak, empedans tabanlı yaklaşımlar Cras veya sitotoksik CD8 T hücre işlevselliği ölçmek amacı diğer yaklaşımlar için iyi alternatifler olabilir.

Watch this Scientific Journal Video about Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System at JoVE.com

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

Krom salınım tahlili, sitotoksik T hücresi aktivitesini tespit etmek için ortak bir tahlil, bazı sınırlamalar vardır. Bu madde, antijen spesifik CD8 T hücreleri ve insan göğüs kanseri tümör hattı ile, SKBR3 kullanarak, bir empedans dayalı bir yaklaşım hücre öldürme saptama yeteneğine için incelenmiştir.

XCELLigence Sistemi Cr51 Yayın Assay Karşılaştırılması tarafından İnsan Her2neu Özgül CD8 T hücrelerinin Optimal Sitotoksik Aktivite Belirlenmesi

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 ...

determining-optimal-cytotoxic-activity-human-her2neu-specific-cd8-t

Research

JoVE Journal

Immunology and Infection

24.9K Görüntüleme.  College of Medicine, Mayo Clinic. Krom salınım tahlili, sitotoksik T hücresi aktivitesini tespit etmek için ortak bir tahlil, bazı sınırlamalar vardır. Bu madde, antijen spesifik CD8 T hücreleri ve insan göğüs kanseri tümör hattı ile, SKBR3 kullanarak, bir empedans dayalı bir yaklaşım hücre öldürme saptama yeteneğine için incelenmiştir.

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

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Sitotoksik T Lenfositler Beta Hücre Ölümü aracılığı ile değerlendirin yöntemleri

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

İmmünoloji ve Enfeksiyon

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

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 upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

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

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

Vitro Testi HIV-spesifik CD4 T hücre efektör işlevi üzerinde immünoregülatör yolları etkisini değerlendirmek için

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 ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 Enfekte CD4 Doğuştan Algılama Değerlendirilmesi<sup&gt; +</sup&gt; Bir kullanılması plazmositoid dendritik hücreler tarafından T Hücreleri<em&gt; Ex vivo</em&gt; Co-kültür sistemi.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Periferik Kan İnsan alloantijen-özgü T hücrelerinin üretilmesi

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

Sitokin yakalama Sistemi kullanılarak Klinik Uygulamalar için Sitomegalovirüs özgü otomatik Hücre Zenginleştirme T hücreleri

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

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

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Otoimmünite karşı Kök Hücre türetilmiş antijen-özgü düzenleyici T hücrelerinin gelişimini

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

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

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

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

Maymun bağışıklık eksikliği virüsüne özgü CD8 analizi<sup&gt; +</sup&gt; Peptid MHC-I tetramer boyama ile Rhesus macaque maymunlarının T-hücrelerinin

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

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

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

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

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

Situ MHC-tetramer boyama ve kantitatif fenotip antijen spesifik CD8 T hücre dokularında, bereket ve konumunu belirlemek için analiz

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

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Tümör-spesifik CD8+ T hücre tepkiler Immunodominance çalışması Için In vivo sitotoksisite assays terzilik

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...