Melanoma-derived Wnt5a conditions dendritic cells to promote regulatory T cell differentiation via the upregulation of indoleamine 2,3-dioxygenase: novel pharmacological strategies for augmenting immunotherapy efficacy

Abstract

Previous studies have shown the b-catenin signaling path-way to promote the development of tolerogenic dendritic cells (DCs) that are capable of driving regulatory T cell (Treg) differentiation. Interestingly, tolerogenic DCs have recently been described to play a role in carcinogenesis. However, the molecular mechanisms underlying the estab-lishment of immune tolerance by this DC population are poorly understood and the methods by which developing Figure 1 Wnt3a and Wnt5a upregulate IDO expression and activity in DCs. A. Wnt5a induces durable IDO expression by BMDCs based on Western blot analysis at 24 and 48 hrs. Representative of 3 independent experiments. UT, untreated. XAV939, b-catenin inhibitor. B. Both Wnt3a and Wnt5a activate the IDO promoter in COS7 cells transiently transfected with a IDO prom -luciferase reporter plasmid. Performed in triplicate. Representative of 2 independent experiments. P value based on a one-way ANOVA. C. Wnt5a induces DC-derived IDO enzymatic activity based on kynurenine detection by HPLC. Cumulative data from 3 separate experiments. P value based on one-way ANOVA. D. Wnt3a and Wnt5a Stimulate b-catenin Activity in Primary TCF/Lef1-EGFP DCs in vitro. Representative of 2 independent experiments. E. Wnt3a and Wnt5a Induce b-catenin-IDO promoter binding in Primary DCs. Performed after 24 hrs of stimulation. IFN-g induction of STAT1 binding to the IDO promoter serves as positive control. Bottom, Western blot generated from same samples. *P < 0.05. P value based on one-way ANOVA. F. b-catenin immunofluorescence microscopy confirms differential kinetics of Wnt3a-and Wnt5a-induced signaling in DCs. Bottom, examples of b-catenin nuclear translation in response to Wnt3a and Wnt5a (100x). 10 fields counted in triplicate. Representative of 2 independent experiments. All data is mean ± SEM.