Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number R01CA174385. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Reagents Preparation
<ol>
 <li>Prepare RPMI-PLGH by mixing 500 ml RPMI-1640 and 5 ml each of Penicillin-streptomycin, L-glutamine, and HEPES solution.</li>
 <li>Prepare R10 solution by mixing RPMI-PLGH with 10% Fetal Bovine Serum (FBS). The FBS is heat-inactivated at 56 &deg;C for 30 min prior to addition.</li>
 <li>Pre-warm at 37 &deg;C 50 ml of RPMI-PLGH, 15 ml of PBS, and 15 ml of R10 in sterile conical tubes.</li>
 <li>Fabrication of arrays of nanowells in PDMS: The silicon master is fabricated using photolithog...

<ol>
	<li>Louis, C. U., 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=Antitumor+activity+and+long-term+fate+of+chimeric+antigen+receptor-positive+T+cells+in+patients+with+neuroblastoma.">Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma.</a> Blood. 118, 6050-6056 (2011).</li><li>Savoldo, 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=CD28+costimulation+improves+expansion+and+persistence+of+chimeric+antigen+receptor-modified+T+cells+in+lymphoma+patients.">CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients.</a> J. Clin. Invest. 121, 1822-1826 (2011).</li><li>Kalos, 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=T+cells+with+chimeric+antigen+receptors+have+potent+antitumor+effects+and+can+establish+memory+in+patients+with+advanced+leukemia.">T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.</a> Sci. Transl. Med. 3, 95ra73 (2011).</li><li>Porter, D. L., Levine, B. L., Kalos, M., Bagg, A., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+Antigen+Receptor-Modified+T+Cells+in+Chronic+Lymphoid+Leukemia.">Chimeric Antigen Receptor-Modified T Cells in Chronic Lymphoid Leukemia.</a> N. Engl. J. Med. , (2011).</li><li>Restifo, N. P., Dudley, M. E., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+immunotherapy+for+cancer:+harnessing+the+T+cell+response.">Adoptive immunotherapy for cancer: harnessing the T cell response.</a> Nat. Rev. Immunol. 12, 269-281 (2012).</li><li>Pittet, M. J., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging.">Intravital imaging.</a> Cell. 147, 983-991 (2011).</li><li>Sumen, C., Mempel, T. R., Mazo, I. B., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy:+visualizing+immunity+in+context.">Intravital microscopy: visualizing immunity in context.</a> Immunity. 21, 315-329 (2004).</li><li>Lamoreaux, L., Roederer, M., Koup, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+cytokine+optimization+and+standard+operating+procedure.">Intracellular cytokine optimization and standard operating procedure.</a> Nat. Protoc. 1, 1507-1516 (2006).</li><li>Varadarajan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+single-cell+analysis+of+human+CD8++T+cell+functions+reveals+discordance+for+cytokine+secretion+and+cytolysis.">A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis.</a> J. Clin. Invest. 121, 4322-4331 (2011).</li><li>Ogunniyi, A. O., Story, C. M., Papa, E., Guillen, E., Love, 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=Screening+individual+hybridomas+by+microengraving+to+discover+monoclonal+antibodies.">Screening individual hybridomas by microengraving to discover monoclonal antibodies.</a> Nat. Protoc. 4, 767-782 (2009).</li><li>Streeck, H., Frahm, N., Walker, B. 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No conflicts of interest declared.

We have outlined the protocol for a high-throughput single-cell cytolytic assay enabled via co-incubation of effectors and targets in arrays of nanowells (Figure 1). In addition to throughput a major advantage of the technique is the ability to monitor effector-mediated cytotoxicity against desired target cells without the need for target cell engineering which in turn allows for the use of autologous or matched/primary tumor cells as target cells. The spatial confinement allows the retrieval and ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>RPMI-1640 w/o L-glutamine</td>
 <td>Cellgro</td>
 <td>15-040-CV</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin</td>
 <td>Cellgro</td>
 <td>30-002-CI</td>
 <td>10,000 I.U. Penicillin 10,000 &mu;g/ml Streptomycin</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Cellgro</td>
 <td>25-005-CI</td>
 <td>200 mM solution</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma Aldrich</td>
 <td>H3537</td>
 <td>1M</td>
 </tr>
 <tr>
 <td>Fetal bovine serum (FBS)</td>
 <td>Atlanta Biologicals</td>
 <td>S11150</td>
 <td>Lot tested</td>
 </tr>
 <tr>
 <td>Cell Tracker Red Stain</td>
 <td>Invitrogen</td>
 <td>C34552</td>
 <td>50 &mu;g</td>
 </tr>
 <tr>
 <td>Vybrant DyeCycle Violet Stain</td>
 <td>Invitrogen</td>
 <td>V35003</td>
 <td>5 mM</td>
 </tr>
 <tr>
 <td>SYTOX green Nucleic Acid Stain</td>
 <td>Invitrogen</td>
 <td>S7020</td>
 <td>5 mM</td>
 </tr>
 <tr>
 <td>Annexin V-Alexa Fluor 647</td>
 <td>Invitrogen</td>
 <td>A23204</td>
 <td>500 &mu;l</td>
 </tr>
 <tr>
 <td>Dulbecco's PBS</td>
 <td>Cellgro</td>
 <td>21-031-CV</td>
 <td>500 ml</td>
 </tr>
 <tr>
 <td>Noble agar</td>
 <td>DIFCO</td>
 <td>2M220</td>
 <td>100 g</td>
 </tr>
 <tr>
 <td>Trypan Blue</td>
 <td>Sigma Aldrich</td>
 <td>T8154</td>
 <td>0.4% liquid, sterile filtered</td>
 </tr>
 <tr>
 <td>Hemocytometer</td>
 <td>Hausser Scientifics</td>
 <td>1492</td>
 <td>Bright line</td>
 </tr>
 <tr>
 <td>4-well plate</td>
 <td>Thermo Fisher</td>
 <td>167603</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Harrick Plasma Cleaner</td>
 <td>Harrick Plasma</td>
 <td>PDC-32G</td>
 <td>Basic plasma cleaner</td>
 </tr>
 <tr>
 <td>Observer.Z1</td>
 <td>ZEISS</td>
 <td>&nbsp;</td>
 <td>Fluorescent microscope (works with the three parts below)</td>
 </tr>
 <tr>
 <td>Lambda 10-3</td>
 <td>Sutter Instrument</td>
 <td>&nbsp;</td>
 <td>Filter controller</td>
 </tr>
 <tr>
 <td>Lambda DG-4</td>
 <td>Sutter Instrument</td>
 <td>&nbsp;</td>
 <td>Ultra-High-Speed Wavelength switcher</td>
 </tr>
 <tr>
 <td>Hamamatsu EM-CCD Camera</td>
 <td>Hamamatsu</td>
 <td>C9100-13</td>
 <td>CCD-Microscope camera</td>
 </tr>
 <tr>
 <td>15 ml conical tube</td>
 <td>BD Falcon</td>
 <td>352097</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 ml conical tube</td>
 <td>VWR</td>
 <td>3282-345-300</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nikon Biostation</td>
 <td>Nikon Instruments Inc.</td>
 <td>Biostation IM</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass bottom culture dish</td>
 <td>MatTek Corporation</td>
 <td>P35G-0</td>
 <td>35 mm petri dish, 10 mm microwell</td>
 </tr>
 </tbody>
</table>

quantitative high-throughput single-cell cytotoxicity assay for t cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

An example of the application of the high-throughput cytolytic assay is demonstrated in Figure 2. Briefly, labeled CD19-specific CAR+ T cells were co-incubated with labeled mouse EL4 target cells in the individual wells of a nanowell array (Sections 1-5). An initial image was recorded on the automated fluorescent microscope to identify the occupancy (effectors and/or targets) of every single nanowell on the array (Section 6). Image processing was used to identify all nanowells containing exact...

Watch this Scientific Journal Video about Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells at JoVE.com

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

quantitative-high-throughput-single-cell-cytotoxicity-assay-for-t

Research

JoVE Journal

Immunology and Infection

14.9K visualizações.  University of Houston. Descrevemos uma única célula ensaio de elevado débito para medir a citotoxicidade de células T, quando incubado com as células-alvo tumorais. Este método emprega uma matriz elastomérica denso, de sub-nanolitro poços (~ 100000 poços matriz /) para confinar espacialmente as células T e as células alvo em proporções definidas e está acoplado a microscopia de fluorescência para monitorizar alvo efetuador conjugação e apoptose subsequente.