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Ex vivo conditioning with IL-12 protects tumor-infiltrating CD8+ T cells from negative regulation by local IFN-γ

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Abstract

Optimal ex vivo expansion protocols for adoptive cell therapy (ACT) must yield T cells able to effectively home to tumors and survive the inhospitable conditions of the tumor microenvironment (TME), while simultaneously exerting persistent anti-tumor effector functions. Our previous work has shown that ex vivo activation in the presence of IL-12 can induce optimal expansion of murine CD8+ T cells, thus resulting in significant tumor regression after ACT mostly via sustained secretion of IFN-γ. In this report, we further elucidate the mechanism of this potency, showing that IL-12 additionally counteracts the negative regulatory effects of autocrine IFN-γ. IL-12 not only downregulates PD-1 expression by T cells, thus minimizing the effects of IFN-γ-induced PD-L1 upregulation by tumor stromal cells, but also inhibits IFNγR2 expression, thereby protecting T cells from IFN-γ-induced cell death. Thus, the enhanced anti-tumor activity of CD8+ T cells expanded ex vivo in the presence of IL-12 is due not only to the ability of IL-12-stimulated cells to secrete sustained levels of IFN-γ, but also to the additional capacity of IL-12 to counter the negative regulatory effects of autocrine IFN-γ.

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Abbreviations

EthD-1:

Ethidium homodimer-1

qPCR:

Quantitative polymerase chain reaction

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Funding

This work was supported by National Cancer Institute Grants K01CA134927, R21CA188767 and by a Velosano Pilot Research Award.

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Authors and Affiliations

  1. Cleveland Clinic, Cleveland Clinic Lerner College of Medicine, Cleveland, OH, USA

    Lin Lin

  2. Department of Inflammation and Immunity, Cleveland Clinic, Lerner Research Institute, 9500 Euclid Avenue NE40, Cleveland, OH, 44195, USA

    Patricia Rayman, Paul G. Pavicic Jr., Charles Tannenbaum, Thomas Hamilton, James Finke & C. Marcela Diaz-Montero

  3. Department of Hematology and Medical Oncology, Cleveland Clinic, Taussig Cancer Institute, Cleveland, OH, USA

    Alberto Montero & Marc Ernstoff

  4. Department of Pathology, Cleveland Clinic, Robert J. Tomsich Pathology and Laboratory Medicine Institute, Cleveland, OH, USA

    Jennifer Ko

  5. Department of Plastic Surgery, Dermatology and Plastic Surgery Institute, Cleveland Clinic, Cleveland, OH, USA

    Brian Gastman

Authors
  1. Lin Lin
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  2. Patricia Rayman
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  3. Paul G. Pavicic Jr.
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  4. Charles Tannenbaum
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  5. Thomas Hamilton
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  6. Alberto Montero
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  7. Jennifer Ko
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  9. James Finke
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  10. Marc Ernstoff
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  11. C. Marcela Diaz-Montero
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Contributions

LL performed most of the experiments, analyzed the data, and drafted the manuscript. PR and PGP Jr. assisted and/or performed some of the experiments, generated figures, and revised the manuscript. CT, TH, AM, and JF contributed to the design and interpretation of the experiments, and revised the manuscript. JK, BG, and ME oversaw the consent and collection of human samples, contributed to the design and analysis of the experiments, and revised the manuscript. CMD-M coordinated the design and execution of the experiments, data analysis, and manuscript drafting and revision.

Corresponding author

Correspondence to C. Marcela Diaz-Montero.

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Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All animals were housed under specific pathogen-free conditions in accordance with Institutional and Federal guidelines at the Cleveland Lerner Research Institution and the experiments were approved by the local Institutional Animal Care and Use Committee (IACUC). Human samples were collected under IRB-approved tissue collection protocol number 3164.

Informed consent

Informed consent was obtained prior to sample collection.

Animal source

C57BL/6 (Thy1.1−), Pmel-1 transgenic (Thy1.1+Vβ13+), IFNγR1 knockout mice were purchased from Jackson Laboratory (Bar Harbor, ME).

Cell line authentication

B16-F10 cells, derived from a gp100+ spontaneous murine melanoma cell line, were obtained from American Type Culture Collection (ATCC) (Manassas, VA). Cell culture was performed under standardized protocols to ensure that phenotypically similar cells are implanted during each experiment. In our laboratory, cell lines are routinely tested for pathogens, and expanded to produce stock aliquots frozen at the same passage.

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Lin, L., Rayman, P., Pavicic, P.G. et al. Ex vivo conditioning with IL-12 protects tumor-infiltrating CD8+ T cells from negative regulation by local IFN-γ. Cancer Immunol Immunother 68, 395–405 (2019). https://doi.org/10.1007/s00262-018-2280-3

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  • DOI: https://doi.org/10.1007/s00262-018-2280-3

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