Characterization of the in vivo immune network of IDO, tryptophan metabolism, PD-L1, and CTLA-4 in circulating immune cells in melanoma

Abstract

In melanoma, both the induction of immunosuppression by tumor cells and the inflammatory antitumor response can induce an upregulation of counter-regulatory mechanisms such as indoleamine 2,3-dioxygenase (IDO), programmed death-ligand 1 (PD-L1) and CTLA-4⁺ regulatory T-cells (Tregs) in the tumor microenvironment. Even though these immunosuppressive mediators are targets for immunotherapy, research investigating their expression in the peripheral blood is lacking. We therefore, performed flow cytometry on PBMCs of stage I-IV melanoma patients. IDO expression was detected in plasmacytoid dendritic cells (pDC) and monocytic myeloid-derived suppressor cells (mMDSC), and increased in advanced disease stage (p = 0.027). Tryptophan breakdown confirmed the functional activity of IDO and was linked with increased PD-L1+ cytotoxic T-cells (p = 0.009), relative lymphopenia (p = 0.036), and a higher mDC/pDC ratio (p = 0.002). High levels of circulating PD-L1+ cytotoxic T-cells were associated with increased CTLA-4 expression by Tregs (p = 0.005) and MDSC levels (p = 0.033). This illustrates that counter-regulatory immune mechanisms in melanoma should be considered as one interrelated signaling network. Moreover, both increased PD-L1+ T-cells and CTLA-4 expression in Tregs conferred a negative prognosis, indicating their in vivo relevance. Remarkably, circulating CTLA-4, IDO, and pDC levels were altered according to prior invasion of the sentinel lymph node and IDO expression in the sentinel was associated with more IDO+ PBMCs. We conclude that the expression of IDO, PD-L1, and CTLA-4 in the peripheral blood of melanoma patients is strongly interconnected, associated with advanced disease and negative outcome, independent of disease stage. Combination treatments targeting several of these markers are therefore likely to exert a synergistic response.

Keywords: AJCC; American Joint Committee on Cancer system; CC, correlation coefficientCTLA-4; Cytotoxic T Lymphocyte-Associated Antigen 4; DC, dendritic cells; HR, hazard ratio; IDO, indoleamine 2, 3-dioxygenase; IFNγ, interferon-gamma; IQR, interquartile range; Kyn, kynurenine; MDSC, myeloid-derived suppressor cells; MFI, mean fluorescence intensity; OS, overall survival; PBMC, peripheral blood mononuclear cells; PD-1, programmed cell death protein 1; PD-L1, Programmed-Death Ligand 1; Treg, regulatory T-cell; Tryp, tryptophan; UPLC, ultra-performance liquid chromatography; cytotoxic T lymphocyte-associated antigen 4 (CTLA-4); indoleamine 2-3-dioxygenase (IDO); mDC, myeloid DC; mMDSC, monocytic MDSC; melanoma; negative feedback mechanism; pDC, plasmacytoid DC; pmnMDSC, polymorphonuclear MDSC; prognosis; programmed-death ligand 1 (PD-L1); regulatory T-cells.

Figure 1.

Impact of PD-L1+ cytotoxic T-cells and CTLA-4+ Tregs on overall survival. Cox regression analysis of overall survival according to the levels of circulating PD-L1+ cytotoxic T-cells (A) and CTLA-4 expression level of regulatory T-cells (B), after adjustment for disease stage. Data are presented as percentages of live peripheral blood mononuclear cells (PBMCs).