Oxidative stress modulates expression of immune checkpoint genes via activation of AhR signaling

Reactive oxygen species (ROS) are by-products of metabolism of oxygen and they play an important role in normal homeostasis and cell signaling, as well as in the initiation of diseases including cancer when their production is upregulated. Thus, it is imperative to understand the cellular and molecular basis by which ROS impact on various biological and pathological processes. In this report, we show that human keratinocyte cell line (HaCaT) treated with hydrogen peroxide displayed an increased activity of AhR, leading to enhanced expression of its downstream targets including cytochrome P450 genes. Intriguingly, preincubation of the complete culture medium with hydrogen peroxide accelerated AhR activation and its downstream signaling. Subsequent mass spectrometric analysis reveals that the oxidant elicits the production of oxindole, a tryptophan catabolic product. We further demonstrate that 2-oxindole (a major form of oxindole) is capable of activating AhR, strongly suggesting that ROS may exert a significant impact on AhR signaling. Consistent with this, we also observe that hexavalent chromium [Cr(VI)], a heavy metal known to generate ROS in vivo, enhances AhR protein levels, as well as stimulates expression of CYP1A2 in an AhR-dependent manner. Significantly, we show that hydrogen peroxide and 2-oxindole induce expression of IDO1 and PD-L1, two immune checkpoint proteins. Given the role of IDO1 and PD-L1 in mediating T cell activity and/or differentiation, we postulate that ROS in the tumor microenvironment may play a crucial role in immune suppression via perturbing AhR signaling.