Elsevier

Surgery

Volume 163, Issue 3, March 2018, Pages 627-632
Surgery

Inhibition of Interleukin-10 in the tumor microenvironment can restore mesothelin chimeric antigen receptor T cell activity in pancreatic cancer in vitro

https://doi.org/10.1016/j.surg.2017.10.056Get rights and content

Abstract

Background

Pancreatic cancer cells are known to shield themselves from immunosurveillance by secreting immune inhibitory cytokines such as Interleukin-10. Using mesothelin, a differentiating antigen that is overexpressed in pancreatic cancer, we assessed the negative effect of the tumor microenvironment on chimeric antigen receptor T cell–based immunotherapy and its reversal via depletion of Interleukin-10.

Methods

T cells cultured in pancreatic cancer–cell-conditioned medium were transduced with lentiviruses encoding mesothelin–chimeric antigen receptor in the presence or absence of anti-Interleukin-10–blocking antibody.

Results

Coculture supernatants of conditioned medium displayed significant inhibition of interferon γ and granzyme B secretion, both of which are crucial for induction of target cell cytotoxicity. In contrast, this inhibition was restored toward baseline when conditioned medium was Interleukin-10– depleted (p < .05 for both interferon γ and granzyme B). In addition, we observed a significant decrease in mesothelin–chimeric antigen receptor T cell–induced cytotoxicity of BxPC-3 target cells in the presence of conditioned medium. Furthermore, we observed a partial blunting of this inhibition when Interleukin-10 was depleted from the conditioned medium.

Conclusion

Substantial reversal of tumor-derived immunosuppression may be achieved by blocking Interleukin-10 in the local microenvironment, allowing for more effective cytotoxicity of mesothelin-engrafted chimeric antigen receptor T cells and enhancing the potential for clinical application.

Section snippets

Cell lines and conditioned medium

We tested 4 human PC cell lines for mesothelin expression (BxPC-3, Capan-1, PANC-1, and MIA PaCa-2), all of which were positive. Although all were positive, BxPC-3 had the greatest expression and was the only nonmetastatic cell line. Because cancer cell lines derived from earlier-stage and lower-grade disease are better models in which to study the effects of novel therapies and establish proof of principle,15 we chose not to include metastatic tumor cell lines in this initial study and could

Efficiency of CAR engraftment of T cells and lentiviral transduction

Third-generation, mesothelin-CAR vector was constructed using cell-surface scFv linked to the CD8α hinge and transmembrane region. This was connected intracellularly with the cytoplasmic portion of two costimulatory domains, CD28 and 4-1BB (CD137), as well as an activation domain derived from the cytoplasmic part of CD3ζ (Fig 1, A). The CAR construct was incorporated into lentiviral vectors driven by the promoter for elongation factor 1 alpha (EF1α), which is preferable for expression in T

Discussion

The PC microenvironment represents an immunosuppressed state, particularly in the more advanced stages of the disease, because tumor cells in vivo secrete spontaneously protective anti-inflammatory cytokines, such as TGF-β and IL-10, that can specifically defend against immune attack.13 Use of mesothelin-engrafted CAR T cells for PC immunotherapy is an active area of research,12, 19, 20 but immune inhibitory mechanisms often observed in solid tumors, which may compromise the efficacy of

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  • Cited by (0)

    Supported in part by grants from the Michael and Marian Ilitch Foundation and the Detroit International Research and Education Foundation.

    Presented at the Central Surgical Association and Midwest Surgical Association Combined Annual Meeting, July 30-August 1, 2017, Chicago, IL.

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