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Immunology and Infection

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

Published: February 2nd, 2013

DOI:

10.3791/50058

1Department of Chemical and Biomolecular Engineering, University of Houston , 2Division of Pediatrics, Research Unit 907, University of Texas MD Anderson Cancer Center

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 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.

1. Reagents Preparation

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

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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.......

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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 .......

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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.

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

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