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Tumor-Induced Suppressive CD8+ T Cells: Implications for Cancer Immunotherapy
Lukas W. Pfannenstiel, Ph.D., Brian T. Maybruck, Ph.D., Brian R. Gastman, M.D..
Cleveland Clinic, Cleveland, OH, USA.
The immune system has the potential to be a powerful tool to destroy tumors; however despite ample evidence of endogenous anti-tumor immune responses in many patients, as well as years of immunotherapy development, truly effective immune-based therapies remain out of reach. We have previously shown that co-incubation of normal human T cells with various tumor lines can induce dysfunctional changes characterized by the loss of CD27/CD28 expression, hypo-proliferation upon activation, and the gain of suppressive function in vitro. We also found that this process could be inhibited by IL-7 signaling, primarily through enhancing expression of the pro-survival molecule Mcl-1. In the current study we sought to determine whether a similar process could not only be found in mice, which would allow the use of in vivo tumor models to study this process in the context of a natural tumor microenvironment, but also in tumor-resident T cells collected from human patient specimens.
TC-1 mouse tumor cells were used as a model of HPV+ HNSCC. To model adoptive T cell therapy, T cells from transgenic mice expressing a TCR-beta chain specific for HPV-E7 were used. To induce T cell dysfunction in vitro, TC-1 tumor cells were incubated with purified T cells from C57/BL6 mice in 6 well plates for six hours followed by separation and culture for four days. CD8+ TIL cells were isolated from mouse and human tumors by digestion in collagenase/hyaluronidase followed by magnetic bead purification and sorting by FACS. Suppression was measured by thymidine uptake by responder T cells incubated with suppressor CD8+ T cells at various ratios in anti-CD3/CD28 coated 96-well plates. Surface marker staining was performed using fluorichrome-conjugated antibodies followed by analysis by flow-cytometry. PD-1, Tim-3, and Lag-3 checkpoint inhibitor blockade was performed by the use of purified antibodies both in vivo and in vitro. Exogenous IL-7 was delivered in vivo by i.p. injection.
We show that the process of tumor-induced dysfunction also induces the expression of PD-1 in both human and mouse T cells, and that tumor-exposed mouse T cells are also capable of suppressive function. We further show that the mouse and human tumor microenvironment induces large numbers of PD-1+ CD8+ T cells that are also positive for other negative regulators of T cell function including Tim-3, and that these cells are also suppressive ex vivo. Treatment of mice and T cells with antibody blockade of PD-1 and other negative regulators, as well as treatment with IL-7 cytokine can prevent the induction of suppressive function.
These studies demonstrate that a tumor-derived factor can induce the dysfunctional and suppressive ability of CD8+ T cells. These cells then could contribute to the overall immunosuppressive nature of the tumor microenvironment. Blockade of negative regulators of T cell function and use of IL-7 cytokine can help prevent this process, indicating an additional benefit to the clinical use of these immunotherapies. Future work will examine the signaling mechanisms responsible, especially by evaluating the role of Mcl-1 expression in generation of dysfunctional CD8 T cells in vivo.
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