A Role for Tryptophan-2,3-dioxygenase in CD8 T-cell Suppression and Evidence of Tryptophan Catabolism in Breast Cancer Patient Plasma

Abstract

Tryptophan catabolism is an attractive target for reducing tumor progression and improving antitumor immunity in multiple cancers. Tumor infiltration by CD8 T cells correlates with improved prognosis in triple-negative breast cancer (TNBC) and a significant effort is underway to improve CD8 T-cell antitumor activity. In this study, primary human immune cells were isolated from the peripheral blood of patients and used to demonstrate that the tryptophan catabolite kynurenine induces CD8 T-cell death. Furthermore, it is demonstrated that anchorage-independent TNBC utilizes the tryptophan-catabolizing enzyme tryptophan 2,3-dioxygenase (TDO) to inhibit CD8 T-cell viability. Publicly available data revealed that high TDO2, the gene encoding TDO, correlates with poor breast cancer clinical outcomes, including overall survival and distant metastasis-free survival, while expression of the gene encoding the more commonly studied tryptophan-catabolizing enzyme, IDO1 did not. Metabolomic analysis, using quantitative mass spectrometry, of tryptophan and its catabolites, including kynurenine, in the plasma from presurgical breast cancer patients (n = 77) and 40 cancer-free donors (n = 40) indicated a strong correlation between substrate and catabolite in both groups. Interestingly, both tryptophan and kynurenine were lower in the plasma from patients with breast cancer compared with controls, particularly in women with estrogen receptor (ER)-negative and stage III and IV breast cancer. IMPLICATIONS: This study underscores the importance of tryptophan catabolism, particularly in aggressive disease, and suggests that future pharmacologic efforts should focus on developing drugs that target both TDO and IDO1.

Figure 1:. Increased kynurenine leads to primary human CD8 T cell death, which is reversed by an AhR antagonist.

(A) Representative example of gating strategy for flow cytometric analysis: CD8 T cells were gated based on double positivity for CD8 (PE-Cy7) and CD3 (PerCP-Cy5.5). Cell death was determined by positive staining for the fixable viability stain BV421. Proliferation was measured by CFSE dilution. (B) Cells were activated with CD3 and CD28 antibodies for 5 days in the indicated concentration of kynurenine and CD8 T cell death was determined. Each dot represents cells from one human donor. N=9 donors, mean with standard deviation, one-way ANOVA. (C) Cells were treated with kynurenine as described in (B) and assayed for CD8 T cell proliferation. N=9 donors, one-way ANOVA. (D) Representative images (10× magnification) of CD8 T cells on activation Day 5 that were treated with indicated concentrations of kynurenine and either 10 μM of the AhR antagonist CH-223191 (AhRa) or DMSO. (E) CD8 T cells were activated, treated with 10 μM AhRa or vehicle control (DMSO), and assayed for cell death. N=6, mean with standard deviation, paired t-tests.

Figure 2:. The effect of conditioned media from suspended TNBC cells on primary human CD8 T cells is similar to that of purified kynurenine, and is reversed by TDO2 inhibitor 680C91.

(A) Conditioned media was collected from BT549 TNBC cells grown in attached (ATT) or suspended (SUS) culture conditions for either 24 or 48 hours as indicated. CD8 T cells were isolated from the blood of normal donors and activated for 5 days with CD3 and CD28 antibodies in the conditioned media. Each line represents the response of CD8 T cells from one donor. Cell death was measured as described in Figures 1A and B. N=9, paired t-tests. (B) Representative flow cytometric analysis of CD8 T cells for IFNγ production is shown. (C) IFNγ production by CD8 T cells cultured in conditioned media for 48 hours. N=7, paired t-tests. (D) CD8 T cells were activated in conditioned media and either 0.1 μM 680C91 (to inhibit TDO activity) or vehicle control (DMSO). Each line represents the response of CD8 T cells from one human volunteer donor. N=5, paired t-tests. (E) CD8 T cells were activated in conditioned media and treated with either 10 μM CH-223191 (to inhibit AhR activation) or vehicle control (DMSO). N=5, paired t-tests.

Figure 3:. TDO2 expression correlates with breast cancer outcomes, while expression of the more commonly studied IDO1 does not.

(A) Patients were split into TDO2-high (red, n=699) or TDO2–low (black, n=703) groups based on median TDO2 gene expression. Overall survival (left) and distant metastasis-free survival (right) was plotted using KmPlot.com. (B) Patients were split into IDO1-high (red, n=701) or IDO1–low (black, n=701) groups based on median gene expression, and overall survival and distant-metastasis free survival was plotted. N=1402, hazard ratios are given with 95% confidence intervals, logrank test.

Figure 4:. Tryptophan and kynurenine are reduced in breast cancer plasma.

Ultra-high performance liquid chromatography/mass spectrometry was used to measure tryptophan and kynurenine concentrations and nicotinamide abundance in the plasma from normal women and women with breast cancer. The concentrations of tryptophan and kynurenine correlate significantly and positively in both normal (A) (n=40) and breast cancer patient (B) (n=77) plasma by Pearson and Spearman tests. Comparison of tryptophan (C) and kynurenine (D) concentrations in plasma from normal women, all breast cancer patients in the cohort, and ER+ versus ER-breast cancer patients. Mean with standard deviation, *p<0.05, **p<0.01, one-way ANOVA and Tukey’s multiple comparisons test. Comparison of tryptophan (E) and kynurenine (F) concentrations plotted by breast cancer stage is shown. Mean with standard deviation, one-way ANOVA and Tukey’s multiple comparisons test. (G) Comparison of the ratio of tryptophan (TRP) to nicotinamide between plasma from normal women and women with breast cancer. Mean with standard deviation, **** p<0.0001, unpaired t-test. (H) Comparison of the relative abundance of nicotinamide in the plasma from normal and breast cancer patient plasma. Mean with standard deviation, p=0.11, unpaired t-test.

Figure 5:. Breast cancer patient plasma tryptophan and progression-free survival.

Ultra-high performance liquid chromatography/mass spectrometry was used to measure tryptophan concentrations in the plasma from women with breast cancer. Kaplan-Meyer curves plotting progression-free survival of breast cancer patients with ER+ disease (n=49) (A) or ER-disease (n=28) (B) are shown, where progression was defined by local, regional, or distant recurrence, or patient death in the years after surgery. Logrank test.