TY - CHAP
T1 - Cytotoxic and chemotactic dynamics of NK cells quantified by live-cell imaging
AU - Zhu, Yanting
AU - Shi, Jue
N1 - Funding Information:
We thank Dr. Paul Choi and Dr. Timothy Mitchison (Department of Systems Biology, Harvard Medical School) for the Granzyme B-FRET retroviral reporter construct, and Dr. John Albeck and Dr. Peter Sorger (Department of Systems Biology, Harvard Medical School) for the Caspase 8-FRET reporter and the IMS-RP retroviral vector. The work described and discussed in this article was supported by funding from the Hong Kong Research Grant Council (#C2006-17E, #T12-710/16-R and #12302421) to J. Shi and the “Laboratory for Synthetic Chemistry and Chemical Biology” under the Health@InnoHK Program by the Innovation and Technology Commission of Hong Kong.
Publisher Copyright:
© 2023 Elsevier Inc.
PY - 2023/1/16
Y1 - 2023/1/16
N2 - Natural Killer (NK) cells detect and eliminate virus-infected cells and cancer cells, and are crucial players of the human immune defense system. Although the relevant molecular machineries involved in NK cell activation and NK-target cell interactions are largely known, how their collective signaling modulates the dynamic behaviors of NK cells, e.g., motility and cytotoxicity, and the rate-limiting kinetics involved are still in need of comprehensive investigations. In traditional bulk killing assays, heterogeneity and kinetic details of individual NK-target cell interactions are masked, seriously limiting analysis of the underlying dynamic mechanisms. Here we present detailed protocols of a number of live-cell imaging assays using fluorescent protein reporters and/or a live-cell dye that enable the acquisition of quantitative kinetic data at the single cell level for elucidating the mechanism underlying the interaction dynamics of primary human NK cells and epithelial cancer cells. Moreover, we discuss how the imaging data can be analyzed either alone or in combination to quantify and determine the key dynamic steps/intermediates involved in specific NK cell activity, e.g., NK cell cytotoxic modes and their associated kinetics, and NK cell motility toward different cancer targets. These live-cell imaging assays can be easily adapted to analyze the rate-limiting kinetics and heterogeneity of other cell-cell interaction dynamics, e.g., in T cell function.
AB - Natural Killer (NK) cells detect and eliminate virus-infected cells and cancer cells, and are crucial players of the human immune defense system. Although the relevant molecular machineries involved in NK cell activation and NK-target cell interactions are largely known, how their collective signaling modulates the dynamic behaviors of NK cells, e.g., motility and cytotoxicity, and the rate-limiting kinetics involved are still in need of comprehensive investigations. In traditional bulk killing assays, heterogeneity and kinetic details of individual NK-target cell interactions are masked, seriously limiting analysis of the underlying dynamic mechanisms. Here we present detailed protocols of a number of live-cell imaging assays using fluorescent protein reporters and/or a live-cell dye that enable the acquisition of quantitative kinetic data at the single cell level for elucidating the mechanism underlying the interaction dynamics of primary human NK cells and epithelial cancer cells. Moreover, we discuss how the imaging data can be analyzed either alone or in combination to quantify and determine the key dynamic steps/intermediates involved in specific NK cell activity, e.g., NK cell cytotoxic modes and their associated kinetics, and NK cell motility toward different cancer targets. These live-cell imaging assays can be easily adapted to analyze the rate-limiting kinetics and heterogeneity of other cell-cell interaction dynamics, e.g., in T cell function.
KW - Natural Killer cell
KW - Live-cell imaging
KW - Single cell dynamics
KW - NK-cancer cell interaction
KW - NK cell cytotoxicity
KW - NK cell motility
UR - https://www.sciencedirect.com/bookseries/methods-in-cell-biology/vol/173/suppl/C
UR - http://www.scopus.com/inward/record.url?scp=85138526153&partnerID=8YFLogxK
U2 - 10.1016/bs.mcb.2022.07.006
DO - 10.1016/bs.mcb.2022.07.006
M3 - Chapter
C2 - 36653085
AN - SCOPUS:85138526153
SN - 9780323901543
T3 - Methods in Cell Biology
SP - 49
EP - 64
BT - The Immunological Synapse Part A
A2 - Thomas, Clément
A2 - Galluzzi, Lorenzo
PB - Academic Press Inc.
CY - San Diego
ER -