Identification, sorting and profiling of functional killer cells via the capture of fluorescent target

Nature Biomedical Engineering (2023)Cite this article

10 Altmetric

Metrics details

Assays for assessing cell-mediated cytotoxicity are largely target-cell-centric and cannot identify and isolate subpopulations of cytotoxic effector cells. Here we describe an assay compatible with flow cytometry for the accurate identification and sorting of functional killer-cell subpopulations in co-cultures. The assay, which we named PAINTKiller (for ‘proximity affinity intracellular transfer identification of killer cells’), relies on the detection of an intracellular fluorescent protein ‘painted’ by a lysed cell on the surface of the lysing cytotoxic cell (specifically, on cell lysis the intracellular fluorescein derivative carboxyfluorescein succinimidyl ester is captured on the surface of the natural killer cell by an antibody for anti-fluorescein isothiocyanate linked to an antibody for the pan-leucocyte surface receptor CD45). The assay can be integrated with single-cell RNA sequencing for the analysis of molecular pathways associated with cell cytotoxicity and may be used to uncover correlates of functional immune responses.

This is a preview of subscription content, access via your institution

Access Nature and 54 other Nature Portfolio journals

Get Nature+, our best-value online-access subscription

$29.99 / 30 days

cancel any time

Subscribe to this journal

Receive 12 digital issues and online access to articles

$99.00 per year

only $8.25 per issue

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Curated hallmark gene sets are publicly available from Molecular Signatures Database (MSigDB, v.7.4). Sequencing data from PAINTKiller-seq are available from the GEO database, with accession number GSE207508. All data needed to evaluate the conclusions described in this paper are included within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.

The R code to reproduce the analyses shown in the figures is available on Zenodo at https://doi.org/10.5281/zenodo.8004120.

Kagi, D., Ledermann, B., Burki, K., Zinkernagel, R. M. & Hengartner, H. Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo. Annu. Rev. Immunol. 14, 207–232 (1996).

Article CAS PubMed Google Scholar

Prager, I. & Watzl, C. Mechanisms of natural killer cell-mediated cellular cytotoxicity. J. Leukoc. Biol. 105, 1319–1329 (2019).

Article CAS PubMed Google Scholar

Brunner, K. T., Mauel, J., Cerottini, J. C. & Chapuis, B. Quantitative assay of the lytic action of immune lymphoid cells on 51Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology 14, 181–196 (1968).

CAS PubMed PubMed Central Google Scholar

Sepp, A., Binns, R. M. & Lechler, R. I. Improved protocol for colorimetric detection of complement-mediated cytotoxicity based on the measurement of cytoplasmic lactate dehydrogenase activity. J. Immunol. Methods 196, 175–180 (1996).

Article CAS PubMed Google Scholar

Wong, P., Wagner, J. A., Berrien-Elliott, M. M., Schappe, T. & Fehniger, T. A. Flow cytometry-based ex vivo murine NK cell cytotoxicity assay. STAR Protoc. 2, 100262 (2021).

Article CAS PubMed PubMed Central Google Scholar

Kandarian, F., Sunga, G. M., Arango-Saenz, D. & Rossetti, M. A flow cytometry-based cytotoxicity assay for the assessment of human NK cell activity. J. Vis. Exp. (126), e56191 (2017).

Wu, X., Zhang, Y., Li, Y. & Schmidt-Wolf, I. G. H. Improvements in flow cytometry-based cytotoxicity assay. Cytometry A 99, 680–688 (2021).

Article CAS PubMed Google Scholar

Peper, J. K. et al. An impedance-based cytotoxicity assay for real-time and label-free assessment of T-cell-mediated killing of adherent cells. J. Immunol. Methods 405, 192–198 (2014).

Article CAS PubMed Google Scholar

Vembadi, A., Menachery, A. & Qasaimeh, M. A. Cell cytometry: review and perspective on biotechnological advances. Front. Bioeng. Biotechnol. 7, 147 (2019).

Article PubMed PubMed Central Google Scholar

Slota, M., Lim, J. B., Dang, Y. & Disis, M. L. ELISpot for measuring human immune responses to vaccines. Expert Rev. Vaccines 10, 299–306 (2011).

Article CAS PubMed PubMed Central Google Scholar

Alter, G., Malenfant, J. M. & Altfeld, M. CD107a as a functional marker for the identification of natural killer cell activity. J. Immunol. Methods 294, 15–22 (2004).

Article CAS PubMed Google Scholar

Bryceson, Y. T., March, M. E., Barber, D. F., Ljunggren, H. G. & Long, E. O. Cytolytic granule polarization and degranulation controlled by different receptors in resting NK cells. J. Exp. Med. 202, 1001–1012 (2005).

Article CAS PubMed PubMed Central Google Scholar

Wolint, P., Betts, M. R., Koup, R. A. & Oxenius, A. Immediate cytotoxicity but not degranulation distinguishes effector and memory subsets of CD8+ T cells. J. Exp. Med. 199, 925–936 (2004).

Article CAS PubMed PubMed Central Google Scholar

Skelley, A. M., Kirak, O., Suh, H., Jaenisch, R. & Voldman, J. Microfluidic control of cell pairing and fusion. Nat. Methods 6, 147–152 (2009).

Article CAS PubMed PubMed Central Google Scholar

Dura, B. et al. Profiling lymphocyte interactions at the single-cell level by microfluidic cell pairing. Nat. Commun. 6, 5940 (2015).

Article CAS PubMed Google Scholar

Dura, B. et al. Longitudinal multiparameter assay of lymphocyte interactions from onset by microfluidic cell pairing and culture. Proc. Natl Acad. Sci. USA 113, E3599–E3608 (2016).

Article CAS PubMed PubMed Central Google Scholar

Assenmacher, M., Lohning, M. & Radbruch, A. Detection and isolation of cytokine secreting cells using the cytometric cytokine secretion assay. Curr. Protoc. Immunol. https://doi.org/10.1002/0471142735.im0627s46 (2002).

McKinnon, K. M. Flow cytometry: an overview. Curr. Protoc. Immunol. https://doi.org/10.1002/cpim.40 (2018).

Deng, N. & Mosmann, T. R. Optimization of the cytokine secretion assay for human IL-2 in single and combination assays. Cytometry A 87, 777–783 (2015).

Article CAS PubMed PubMed Central Google Scholar

Trinklein, N. D. et al. Efficient tumor killing and minimal cytokine release with novel T-cell agonist bispecific antibodies. MAbs 11, 639–652 (2019).

Article CAS PubMed PubMed Central Google Scholar

Good, Z. et al. Proliferation tracing with single-cell mass cytometry optimizes generation of stem cell memory-like T cells. Nat. Biotechnol. 37, 259–266 (2019).

Article CAS PubMed PubMed Central Google Scholar

Matera, G., Lupi, M. & Ubezio, P. Heterogeneous cell response to topotecan in a CFSE-based proliferation test. Cytometry A 62, 118–128 (2004).

Article PubMed Google Scholar

Ponchio, L. et al. Mitomycin C as an alternative to irradiation to inhibit the feeder layer growth in long-term culture assays. Cytotherapy 2, 281–286 (2000).

Article CAS PubMed Google Scholar

Llames, S., Garcia-Perez, E., Meana, A., Larcher, F. & del Rio, M. Feeder layer cell actions and applications. Tissue Eng. B Rev. 21, 345–353 (2015).

Article CAS Google Scholar

Krzewski, K., Gil-Krzewska, A., Nguyen, V., Peruzzi, G. & Coligan, J. E. LAMP1/CD107a is required for efficient perforin delivery to lytic granules and NK-cell cytotoxicity. Blood 121, 4672–4683 (2013).

Article CAS PubMed PubMed Central Google Scholar

Liu, D. et al. Integrin-dependent organization and bidirectional vesicular traffic at cytotoxic immune synapses. Immunity 31, 99–109 (2009).

Article CAS PubMed PubMed Central Google Scholar

Suen, W. C., Lee, W. Y., Leung, K. T., Pan, X. H. & Li, G. Natural killer cell-based cancer immunotherapy: a review on 10 years completed clinical trials. Cancer Invest. 36, 431–457 (2018).

Article CAS PubMed Google Scholar

Surman, D. R., Dudley, M. E., Overwijk, W. W. & Restifo, N. P. Cutting edge: CD4+ T cell control of CD8+ T cell reactivity to a model tumor antigen. J. Immunol. 164, 562–565 (2000).

Article CAS PubMed Google Scholar

Rosenberg, S. A. & Dudley, M. E. Cancer regression in patients with metastatic melanoma after the transfer of autologous antitumor lymphocytes. Proc. Natl Acad. Sci. USA 101, 14639–14645 (2004).

Article CAS PubMed PubMed Central Google Scholar

Caligiuri, M. A. Human natural killer cells. Blood 112, 461–469 (2008).

Article CAS PubMed PubMed Central Google Scholar

Spiegel, J. Y. et al. CAR T cells with dual targeting of CD19 and CD22 in adult patients with recurrent or refractory B cell malignancies: a phase 1 trial. Nat. Med. 27, 1419–1431 (2021).

Article CAS PubMed PubMed Central Google Scholar

Prager, I. et al. NK cells switch from granzyme B to death receptor-mediated cytotoxicity during serial killing. J. Exp. Med. 216, 2113–2127 (2019).

Article CAS PubMed PubMed Central Google Scholar

Park, J. et al. Multifunctional microparticles with stimulation and sensing capabilities for facile NK cell activity assay. ACS Sens. 6, 693–697 (2021).

Article CAS PubMed Google Scholar

Dybkaer, K. et al. Genome wide transcriptional analysis of resting and IL2 activated human natural killer cells: gene expression signatures indicative of novel molecular signaling pathways. BMC Genomics 8, 230 (2007).

Article PubMed PubMed Central Google Scholar

Zhang, J. et al. Sequential actions of EOMES and T-BET promote stepwise maturation of natural killer cells. Nat. Commun. 12, 5446 (2021).

Article CAS PubMed PubMed Central Google Scholar

van Helden, M. J. et al. Terminal NK cell maturation is controlled by concerted actions of T-bet and Zeb2 and is essential for melanoma rejection. J. Exp. Med. 212, 2015–2025 (2015).

Article PubMed PubMed Central Google Scholar

Chester, C., Fritsch, K. & Kohrt, H. E. Natural killer cell immunomodulation: targeting activating, inhibitory, and co-stimulatory receptor signaling for cancer immunotherapy. Front. Immunol. 6, 601 (2015).

Article PubMed PubMed Central Google Scholar

Connor, J. H., Weiser, D. C., Li, S., Hallenbeck, J. M. & Shenolikar, S. Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1. Mol. Cell. Biol. 21, 6841–6850 (2001).

Article CAS PubMed PubMed Central Google Scholar

Wang, X. W. et al. GADD45 induction of a G2/M cell cycle checkpoint. Proc. Natl Acad. Sci. USA 96, 3706–3711 (1999).

Article CAS PubMed PubMed Central Google Scholar

Li, Y., Yu, M., Yin, J., Yan, H. & Wang, X. Enhanced calcium signal induces NK cell degranulation but inhibits its cytotoxic activity. J. Immunol. 208, 347–357 (2022).

Article CAS PubMed Google Scholar

Jacobi, C. et al. Exposure of NK cells to intravenous immunoglobulin induces IFN gamma release and degranulation but inhibits their cytotoxic activity. Clin. Immunol. 133, 393–401 (2009).

Article CAS PubMed Google Scholar

Stoeckius, M. et al. Simultaneous epitope and transcriptome measurement in single cells. Nat. Methods 14, 865–868 (2017).

Article CAS PubMed PubMed Central Google Scholar

Parish, C. R. Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol. Cell Biol. 77, 499–508 (1999).

Article CAS PubMed Google Scholar

Zheng, Y., Tang, L., Mabardi, L., Kumari, S. & Irvine, D. J. Enhancing adoptive cell therapy of cancer through targeted delivery of small-molecule immunomodulators to internalizing or noninternalizing receptors. ACS Nano 11, 3089–3100 (2017).

Article CAS PubMed PubMed Central Google Scholar

Wu, T., Womersley, H. J., Wang, J. R., Scolnick, J. & Cheow, L. F. Time-resolved assessment of single-cell protein secretion by sequencing. Nat. Methods 20, 723–734 (2023).

Article CAS PubMed Google Scholar

Roelli, P., bbimber, Flynn, B., santiagorevale & Gui, G. Hoohm/CITE-seq-Count: 1.4.2 (1.4.2). Zenodo https://doi.org/10.5281/zenodo.2585469 (2019).

Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184, 3573–3587.e29 (2021).

Article CAS PubMed PubMed Central Google Scholar

Pont, F., Tosolini, M. & Fournie, J. J. Single-Cell Signature Explorer for comprehensive visualization of single cell signatures across scRNA-seq datasets. Nucleic Acids Res. 47, e133 (2019).

Article CAS PubMed PubMed Central Google Scholar

Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284–287 (2012).

Article CAS PubMed PubMed Central Google Scholar

Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).

Article CAS PubMed PubMed Central Google Scholar

Download references

We acknowledge technical support from the Flow Cytometry Laboratory (NUS Medical Sciences Cluster) for cell sorting. L.F.C. was supported for the research described in this study by the National Medical Research Council (MOH-OFIRG18nov-003), the Ministry of Education of Singapore (MOE-T2EP30120-0008) and Singapore-MIT Alliance for Research and Technology Critical Analytics for Manufacturing of Personalized-Medicine (SMART CAMP) Interdisciplinary Research Group.

These authors contributed equally: Yen Hoon Luah, Tongjin Wu.

Institute for Health Innovation and Technology, National University of Singapore, Singapore, Singapore

Yen Hoon Luah, Tongjin Wu & Lih Feng Cheow

Critical Analytics for Manufacturing of Personalized-Medicine Interdisciplinary Research Group, Singapore-MIT Alliance in Research and Technology, Singapore, Singapore

Yen Hoon Luah & Lih Feng Cheow

Department of Biomedical Engineering, College of Design and Engineering, National University of Singapore, Singapore, Singapore

Tongjin Wu & Lih Feng Cheow

You can also search for this author in PubMed Google Scholar

L.F.C., Y.H.L. and T.W. conceived and designed the study. T.W. performed the experiments corresponding to Figs. 2, 4 and 7. Y.H.L. performed the experiments corresponding to Fig. 3. T.W. and L.F.C. performed the PAINTKiller-seq data analysis. L.F.C., T.W. and Y.H.L. wrote the manuscript. All authors commented on the manuscript and approved the submission.

Correspondence to Lih Feng Cheow.

The authors declare no competing interests.

Nature Biomedical Engineering thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

a, Unfiltered gene-expression data from two sequencing lanes (‘Exp. 1’ and ‘Exp. 2’). Cells with the following criteria were filtered for downstream analysis: gene numbers > 1,200 and < 8,000, gene-expression counts > 1,000 and < 40,000, mitochondrial genes < 10% of total genes detected. b, The cell samples, including NK-K562 co-culture group (‘NK + K562’), NK only group (‘NK only’) and isotype staining control (‘Isotype ctrl’), were demultiplexed by their staining intensity of anti-β2M hashtags. Cell numbers for each group were shown in bracket.

a, b, Cells were clustered by their transcriptional profiles (a), and non-NK cells were excluded according to the expression of lineage-associated genes such as CD3E for T cells and HBA1/HBA2 for K562 cells (b). c, NK cells were re-grouped by their transcriptional profiles. A total of 13 clusters were identified. Cluster 6 and 7 were mostly found in NK sample that has been co-cultured with K562 cells.

Supplementary figures and tables.

Source data.

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

Luah, Y.H., Wu, T. & Cheow, L.F. Identification, sorting and profiling of functional killer cells via the capture of fluorescent target-cell lysate. Nat. Biomed. Eng (2023). https://doi.org/10.1038/s41551-023-01089-z

Download citation

Received: 15 July 2022

Accepted: 04 August 2023

Published: 31 August 2023

DOI: https://doi.org/10.1038/s41551-023-01089-z

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative