Abstract B55: Suppression of CD8 T-cell activation by tryptophan catabolism in triple-negative breast cancer
Authors: Greene Lisa I. , Bruno Tullia C. , Rogers Thomas J. , Slansky Jill E. , Borges Virginia F. , Richer Jennifer K. 
Published: May 2016

Background: The ability of cancer cells to evade and suppress an antitumor immune response is increasingly recognized as a critical feature of aggressive, metastatic disease. Conversion of the amino acid tryptophan (trp) into the catabolite kynurenine (kyn) and the subsequent secretion of kyn is a current area of interest in the field of antitumor immunity, given that trp depletion is a trigger for T-cell death, and that kyn binds to and activates the aryl-hydrocarbon receptor (AhR) in immune cells, often with immunosuppressive consequences. In a screen to identify mechanisms promoting survival of triple-negative breast cancer (TNBC) cells in transit during the metastatic cascade, our lab discovered that trp-to-kyn catabolism is significantly increased by TNBC cells in forced-suspension culture due to upregulation of the gene encoding the rate-limiting trp-catabolizing enzyme tryptophan 2,3-dioxygenase (TDO2).

Hypothesis: In addition to promoting survival of TNBC cells during metastasis, TDO2-dependent trp-to-kyn catabolism may facilitate tumor-mediated immune suppression, further contributing to the aggressive nature of this breast cancer subtype.

Methods: TNBC cell lines were plated in adherent or forced-suspension culture for 24 hours, using poly (2-hydroxyethyl methacrylate)-coated plates to prevent adhesion. TDO2 mRNA (using quantitative PCR) and protein (immunoblotting) were measured in adherent versus suspended cells. Secreted kyn was measured using liquid chromatography-mass spectrometry and inhibited with a selective TDO2 inhibitor, 680C91. To test the effect of kyn on CD8 T-cell function, primary human CD8 T-cells were isolated from the blood of healthy human donors using a positive selection kit. T-cell proliferation was measured by dilution of the cell-permeable dye carboxyfluorescein succinimidyl ester and T-cell death was measured by incorporation of a fixable viability dye. Flow cytometry was used to measure both surface and intracellular T-cell proteins.

Results: The rate limiting enzyme TDO2 was upregulated in BT549 cells in suspension by 20.1-fold at the mRNA level and 5.4-fold at the protein level. Secreted kyn increased by 2.4-fold in suspension culture (0.46 μM secreted kyn by adherent cells, 1.09 μM kyn secreted by suspended cells, n=3 replicates), and this increase was reversed by addition of the TDO2 inhibitor 680C91 (p&lt;0.0001). Over the course of a five day CD3/CD28 activation, purified kyn reduced CD8 T-cell proliferation and increased CD8 T-cell death in a dose-dependent manner (n=6-9 donors, p&lt;0.05 at 25, 50, and 100 μM kyn), and these effects were partially reversed by the addition of an AhR antagonist (n=6 donors, p&lt;0.05 at 25 μM kyn). Kyn also reduced expression of granzyme B by live CD8 T-cells at high doses (n=4 donors, p=0.05 at 100 μM kyn), but did not alter expression of perforin or tumor necrosis factor alpha. Consistent with these results, culture of human CD8 T-cells in conditioned media from suspended BT549 cells over the course of a five day CD3/CD28 activation resulted in reduced proliferation and increased cell death in CD8 T-cells (n=6 donors, p&lt;0.05) compared to culture in conditioned media from adherent BT549 cells. Expression of the pro-inflammatory cytokine interferon gamma by CD8 T-cells cultured in conditioned media from suspended BT549 cells may also be reduced (n=4 donors, p=0.08).

Conclusions: Together, these results suggest a role for TNBC-mediated trp-to-kyn catabolism in suppressing cytotoxic T-cells. Further in vivo studies will indicate whether TDO2 activity might be a rational therapeutic target for improving antitumor immunity in patients with this aggressive subtype of breast cancer.

Citation Format: Lisa I. Greene, Tullia C. Bruno, Thomas J. Rogers, Jill E. Slansky, Virginia F. Borges, Jennifer K. Richer. Suppression of CD8 T-cell activation by tryptophan catabolism in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B55.