Cancer Immunology, Immunotherapy

, Volume 60, Issue 5, pp 739–749

In vitro generated anti-tumor T lymphocytes exhibit distinct subsets mimicking in vivo antigen-experienced cells

  • Shicheng Yang
  • Gattinoni Luca
  • Fang Liu
  • Yun Ji
  • Zhiya Yu
  • Nicholas P. Restifo
  • Steven A. Rosenberg
  • Richard A. Morgan
Original article

DOI: 10.1007/s00262-011-0977-7

Cite this article as:
Yang, S., Luca, G., Liu, F. et al. Cancer Immunol Immunother (2011) 60: 739. doi:10.1007/s00262-011-0977-7

Abstract

The T-lymphocyte pool can be subdivided into naïve (Tn), effector memory (Tem), and central memory (Tcm) T cells. In this study, we characterized in vitro short-term cultured anti-tumor human T lymphocytes generated by lentiviral transduction with an anti-tumor antigen TCR vector. Within 2 weeks of in vitro culture, the cultured T cells showed a Tcm-like phenotype illustrated by a high percentage of CD62L and CD45RO cells. When the cells were sorted into populations that were CD45RO+/CD62L-(Tem), CD45RO+/CD62L+(Tcm), or CD45ROlow/CD62L+(Tn) and co-cultured with antigen-matched tumor lines, the magnitude of cytokine release from these populations for IFNγ (Tn < Tcm < Tem) and IL-2 (Tn > Tcm > Tem) mimicked the types of immune cell responses observed in vivo. In comparing cell-mediated effector function, Tn were found to be deficient (relative to Tcm and Tem) in the ability to form conjugates with tumor cells and subsequent lytic activity. Moreover, analysis of the gene expression profiles of the in vitro cultured and sorted T-cell populations also demonstrated patterns consistent with their in vivo counterparts. When Tcm and Tem were tested for the ability to survive in vivo, Tcm displayed significantly increased engraftment and persistence in NOD/SCID/γc−/− mice. In general, a large percentage of in vitro generated anti-tumor T lymphocytes mimic a Tcm-like phenotype (based on phenotype, effector function, and increased persistence in vivo), which suggests that these Tcm-like cultured T cells may be optimal for adoptive immunotherapy.

Keywords

Gene therapy Lentiviral vector T-cell receptor Central memory cells Effector memory cells Tumor immunity 

Supplementary material

262_2011_977_MOESM1_ESM.eps (722 kb)
Sorting of in vitro cultured anti-tumor T lymphocytes for in vivo engraftment. A. Schematic illustration of in vitro transduced and expanded T lymphocytes. PBMC were activated by anti-CD3/CD28 beads. The next day, cells were transduced with lentiviral vector harboring anti-tumor TCR, and 6 h later, the cells were transferred from 6-well plates to 75-cm2 flasks. The cells were maintained for 14 days before harvesting. B. Calibration for sorting and post-evaluation. Upper panel, the percentage of cells defined by square in Tem and Tcm was sorted; middle panel, post-sort re-evaluation; lower panel, the expression of transduced anti-tumor TCR on sorted Tem and Tcm populations was measured by MART-1 Tetramer staining (EPS 721 kb)
262_2011_977_MOESM2_ESM.eps (431 kb)
Summary for generation of anti-tumor T lymphocytes using anti-CD3/CD28 beads activation and lentiviral vector transduction. Twenty million of PBMC per well of 6-well plates from 6 donors were activated by anti-CD3/CD28 beads on day 0 (beads to cells, 2:1), and on day 1 post-stimulation, the cells were transduced with lentiviral vector harboring MART-1 antigen TCR by spinoculation. Six hours post-transduction, the cells were transferred to 75-cm2 culture dishes with a total volume of 30 ml culture medium in horizontal position, and the cell density was maintained below 1.5 × 106/ml in culture for 14 days. The phenotype of transduced PBMC at day 14 was analyzed using a panel of antibodies as denoted on top of each FACS image. The fold expansion from each donor was shown on last column of the table (EPS 431 kb)
262_2011_977_MOESM3_ESM.xlt (616 kb)
Gene expression profile of sorted Tem, Tcm, and Tn populations analyzed by Microarray. The total RNA extracted from sorted populations was previously described in Fig. 5. One-way hierarchical clustering of sorted T-cell samples was conducted, and 2-fold differentially expressed genes were chosen for analysis. A set of 597 probe IDs was identified as differentially expressed among cultured T-cell subsets and used for clustering (XLT 616 kb)

Copyright information

© Springer-Verlag (outside the USA) 2011

Authors and Affiliations

  • Shicheng Yang
    • 1
  • Gattinoni Luca
    • 1
  • Fang Liu
    • 1
  • Yun Ji
    • 1
  • Zhiya Yu
    • 1
  • Nicholas P. Restifo
    • 1
  • Steven A. Rosenberg
    • 1
  • Richard A. Morgan
    • 1
  1. 1.Surgery Branch, Center for Cancer Research, National Cancer InstituteNational Institutes of HealthBethesdaUSA

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