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Ex vivo identification, isolation and analysis of tumor-cytolytic T cells

Nature Medicine volume 9, pages 1377–1382 (2003)Cite this article

Abstract

We isolated pure, viable populations of tumor-cytolytic T cells directly from patient blood samples using flow cytometric quantification of the surface mobilization of CD107a—an integral membrane protein in cytolytic granules—as a marker for degranulation after tumor stimulation. We show that tumor-cytolytic T cells are indeed elicited in patients after cancer vaccination, and that tumor reactivity is strongly correlated with efficient T-cell recognition of peptide-bearing targets. We combined CD107a mobilization with peptide–major histocompatibility complex (P-MHC) tetramer staining to directly correlate antigen specificity and cytolytic ability on a single-cell level. This showed that tumor-cytolytic T cells with high recognition efficiency represent only a minority of peptide-specific T cells elicited in patients after heteroclitic peptide vaccination. We were also able to expand these cells to high numbers ex vivo while maintaining their cytolytic potential. These techniques will be useful not only for immune monitoring of cancer vaccine trials, but also for adoptive cellular immunotherapy after ex vivo expansion. The ability to rapidly identify and isolate tumor-cytolytic T cells would be very useful in cancer immunotherapy.

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Figure 1: CD107a mobilization by high– and low–recognition efficiency T-cell clones.
Figure 2: Identification of tumor-reactive T cells from a heterogeneous cell line by CD107a mobilization.
Figure 3: Identification of high–recognition efficiency cytolytic T cells in post-vaccine PBMCs.
Figure 4: High–recognition efficiency cytolytic T cells represent a small fraction of tetramer-positive cells.

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Acknowledgements

This work was supported by National Institutes of Health grant R01 CA 090809 (P.L.) and the Damon Runyon Cancer Research Foundation Scholar Award (P.L.). We thank J. Harvell for immunohistochemical staining of melanoma cell lines, C. Yee for kindly providing the mel526 cell line, J. Yu for antibody conjugation and A. Salles and M. Mallipeddi for technical assistance.

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Authors and Affiliations

  1. Department of Medicine, Stanford University, 269 Campus Drive, Stanford, 94305, California, USA

    Valerie Rubio, Tor B Stuge, Naileshni Singh & Peter P Lee

  2. Laboratory of Immunology, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, Bethesda, 20892, Maryland, USA

    Michael R Betts

  3. Norris Cancer Center, University of Southern California, 1441 Eastlake Avenue, Suite 3454, Los Angeles, 90033, California, USA

    Jeffrey S Weber

  4. ImmunoTechnology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, Bethesda, 20892, Maryland, USA

    Mario Roederer

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  1. Valerie Rubio
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Correspondence to Peter P Lee.

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Rubio, V., Stuge, T., Singh, N. et al. Ex vivo identification, isolation and analysis of tumor-cytolytic T cells. Nat Med 9, 1377–1382 (2003). https://doi.org/10.1038/nm942

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