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Isolation of tumour-reactive lymphocytes from peripheral blood via microfluidic immunomagnetic cell sorting

Nature Biomedical Engineering volume 7, pages 1188–1203 (2023)Cite this article

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Abstract

The clinical use of tumour-infiltrating lymphocytes for the treatment of solid tumours is hindered by the need to obtain large and fresh tumour fractions, which is often not feasible in patients with unresectable tumours or recurrent metastases. Here we show that circulating tumour-reactive lymphocytes (cTRLs) can be isolated from peripheral blood at high yield and purity via microfluidic immunomagnetic cell sorting, allowing for comprehensive downstream analyses of these rare cells. We observed that CD103 is strongly expressed by the isolated cTRLs, and that in mice with subcutaneous tumours, tumour-infiltrating lymphocytes isolated from the tumours and rapidly expanded CD8+CD103+ cTRLs isolated from blood are comparably potent and respond similarly to immune checkpoint blockade. We also show that CD8+CD103+ cTRLs isolated from the peripheral blood of patients and co-cultured with tumour cells dissociated from their resected tumours resulted in the enrichment of interferon-γ-secreting cell populations with T-cell-receptor clonotypes substantially overlapping those of the patients’ tumour-infiltrating lymphocytes. Therapeutically potent cTRLs isolated from peripheral blood may advance the clinical development of adoptive cell therapies.

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Fig. 1: Non-invasive collection of tumour-reactive cells in blood circulation for cancer immunotherapy.
Fig. 2: Isolation of tumour-reactive TRLs in blood circulation.
Fig. 3: Molecular and phenotypic signature of cTRL during and post migration.
Fig. 4: cTRLs exhibit significant levels of activity against primary and metastasized tumours in murine models.
Fig. 5: Synergistic effects of cTRLs and ICB/co-stimulatory molecules.
Fig. 6: CD103+ defines cTRL population in human PBMC.
Fig. 7: CD8+CD103+ cTRLs are phenotypically and clonally tumour specific.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. The RNA-seq data are available from the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) under the access code GSE227345. The unprocessed TCR-sequencing files and CyTOF data are too large to be publicly shared, yet they are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank T. Chen at the Centre for Advanced Single Cell Analysis (CASCA), Sick Children Hospital, Toronto for her help in CyTOF, N. Simard at the centralize flow cytometry facility at Temerty Faculty of Medicine, University of Toronto for her help in FACS sorting, A. C. Zhou at the Medicine by Design initiative at the University of Toronto for her comments, W. Xiao and A. Archila at the University Health Network (UHN) for their help in tail vein injection, M. Peralta at the UHN PRP facility and N. Law at the UHN STTARR facility for their help in IHC, J. Jonkman at the UHN AOMF facility for his help in image quantitation, and J. Wei and J. Moffat at the Terrence Donnelly Centre, University of Toronto for donating CT26HA and OT-1 cells. This study was supported in part by the Canadian Institutes of Health Research (grant no. FDN-148415) and the Collaborative Health Research Projects program (CIHR/NSERC partnered). This research was part of the University of Toronto’s Medicine by Design initiative, which receives funding from the Canada First Research Excellence Fund. The study was also supported in part by the McCormick Catalyst Fund at Northwestern University.

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, McCormick School of Engineering, Northwestern University, Evanston, IL, USA

    Zongjie Wang, Sharif Ahmed & Shana O. Kelley

  2. Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada

    Zongjie Wang, Licun Wu, Fatemeh Bavaghar-Zaeimi & Marc de Perrot

  3. Department of Chemistry, Weinberg College of Arts & Sciences, Northwestern University, Evanston, IL, USA

    Mahmoud Labib & Shana O. Kelley

  4. Peninsula Medical School, Faculty of Health, University of Plymouth, Plymouth, UK

    Mahmoud Labib

  5. Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada

    Hansen Wang, Soraly Blanco & Shana O. Kelley

  6. Division of Thoracic Surgery, Toronto General Hospital, University Health Network, Toronto, Ontario, Canada

    Fatemeh Bavaghar-Zaeimi, Nastaran Shokri, Saraf Karim, Kasia Czarnecka-Kujawa & Marc de Perrot

  7. The Edward S. Rogers Sr. Department of Electrical & Computer Engineering, University of Toronto, Toronto, Ontario, Canada

    Edward H. Sargent

  8. Department of Immunology, Division of Translational Immuno-Oncology, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA

    A. J. Robert McGray

  9. Department of Immunology, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada

    Marc de Perrot

  10. International Institute for Nanotechnology, Northwestern University, Evanston, IL, USA

    Shana O. Kelley

  11. Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Shana O. Kelley

  12. Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL, USA

    Shana O. Kelley

  13. Simpson Querrey Institute, Northwestern University, Chicago, IL, USA

    Shana O. Kelley

  14. Chan Zuckerberg Biohub Chicago, Chicago, IL, USA

    Shana O. Kelley

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  1. Zongjie Wang
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Contributions

Z.W. and S.O.K. conceived and designed the experiments. Z.W. performed cell isolation, flow cytometry and CyTOF. S.A. performed the animal study. M.L. performed RNA extraction and qPCR. H.W. extracted the OVA plasmid and assisted with the animal study. L.W. maintained the AE17 cell lines. L.W., F.B.-Z., N.S., S.B. and S.K. managed patient-sample collection, distribution and administration. All authors discussed the results, analysed the data and contributed to the preparation and editing of the manuscript.

Corresponding author

Correspondence to Shana O. Kelley.

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Competing interests

S.O.K. and Z.W. have a filled patent application using parts of the data reported in this article. S.O.K. has a patent ‘Device for capture of particles in a flow’ US10073079 licensed to Cellular Analytics. A.J.R.M. is a paid consultant for Cellular Analytics. M.D.P. received personal fees from Actelion, AstraZeneca, Bayer, Bristol Myers Squibb, Merck and Roche outside of the submitted work. S.O.K. received research funds from Amgen through a sponsored research agreement. The other authors declare no competing interests.

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Nature Biomedical Engineering thanks Rong Fan, Alexandre Harari and Paul Robbins for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Introduction of immunogenic epitopes promote the endogenous immune responses against the inoculated tumors.

a, Workflow of the identification of TRLs and cTRLs via defined epitope models. b, Quantitation of tumor-reactive T cells in tumor and blood. Unpaired t-test, mean ± s.d., each dot represents a biological replicate.

Extended Data Fig. 2 Comparison of the performance of rare cell isolation based on multimer-labeling among FACS, MACS and microfluidics.

Unpaired t-test, mean ± s.d., each dot represents a biological replicate.

Extended Data Fig. 3 Comparison of the rare cTRL sorting based on CD103 labeling among FACS, MACS and microfluidics.

Unpaired t-test, mean ± s.d., each dot represents a biological replicate.

Extended Data Fig. 4 Quantitation of tumor size, survival rate and percentage of infiltrated CD8+ cells in s.c. LLC-1 models in WT C57BL6 mice treated by different populations of lymphocytes (n = 5).

Unpaired t-test, mean ± s.d., each dot represents a biological replicate.

Extended Data Fig. 5 Quantitation of lung metastases in i.v. 4T1 models in nude mice at the endpoint treated by different T cells. (n = 6, 6 layers for each animal L: Lung, T: Tumor).

Unpaired t-test, mean ± s.d., each dot represents a biological replicate.

Extended Data Fig. 6 Quantitation of tumor size, survival rate and percentage of infiltrated CD8+ cells in s.c. MC38 models in RAG−/− C57BL6 mice treated by different therapeutic modalities (n = 5).

Unpaired t-test, mean ± s.d., each dot represents a biological replicate.

Supplementary information

Supplementary Information

Supplementary discussion, methods, figures, tables and references.

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Source data

Source Data

Tumour-growth data for Figs. 4 and 5, and for Extended Data Figs. 4 and 6.

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Wang, Z., Ahmed, S., Labib, M. et al. Isolation of tumour-reactive lymphocytes from peripheral blood via microfluidic immunomagnetic cell sorting. Nat. Biomed. Eng 7, 1188–1203 (2023). https://doi.org/10.1038/s41551-023-01023-3

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