X-ray irradiation triggers via ROS production a canonical Ca2+-dependent immune response in T-lymphocytes

Abstract

For decades X-ray irradiation has been employed in cancer therapy for eliminating malignant cells. In this treatment however not only the tumor cells but also cells in the surrounding tissue and blood are unavoidably exposed to ionizing irradiation (IR). While the cellular and molecular effects of radiation on tumor cells are well studied the impact on the immune cells, which are located coincidentally in the blood vessels and the tumor environment, remain elusive. Because of this still existing knowledge gap the present work aims to uncover the effects of X-ray as a type of IR on immune cells, especially T-lymphocytes in the blood. The primary goal is to reveal early radiation-triggered events and to elucidate their downstream effects. The work is motivated by the idea that an understanding of intracellular reaction mechanisms will help to better assess the risks of radio-therapy for tumor patients. The focus on exposed immune cells bears the possibility that X-ray may generate antagonistic or even agonistic effects in these cells, which will help to improve radiation protocols. In the case of an agonistic, hermetic effect on immune cell it even opens the potential for combining radio-immune therapy for a successful cancer treatment. \nIn previous studies it was shown that ionizing radiation can have beneficial effects on T-lymphocytes and stimulate an immune activation in these cells. In this scenario, redox and calcium signals serve as components in second messenger cascades where they cross react with the canonical signaling events that induce immune activation of T-cells. The latter is initiated by antigen binding to the T-cell receptor (TCR) located on the T-cell surface. This is followed by precisely regulated cytosolic calcium (Ca2+cyt) oscillations, which stimulate the nuclear translocation of the transcription factor nuclear factor of activated T-cells (NFAT) and therefore mediate specific immunological modulations. \nThe data of the current study corroborate results from previous investigations that exposing T-lymphocytes to X-ray doses between 0.1 and 5 Gy triggers an intracellular ROS/Ca2+-dependent signaling cascade in Jurkat T-cells and peripheral blood mononuclear cells (PBMCs). The first resolvable events of the IR-triggered pathway are a transient increase in mitochondrial and cytosolic ROS over the first 1-20 minutes after exposure. This is followed after a lack time, within a large variation of 10-80 min after irradiation by long-lasting (>3 h) Ca2+ oscillations with a highly conserved frequency (˜3 mHz) and a dose-dependent amplitude. The Ca2+ signaling cascade involves a clustering of STIM/Orai proteins forming Ca2+-release activated Ca2+ (CRAC) channels, which in turn activates Ca2+-induced Ca2+-release (CICR). As a downstream consequence of the dynamic Ca2+ changes in the cytosol the transcription factor NFAT is translocating from the cytosol into the nucleus approximately 50 min post radiation exposure. As result of the nuclear NFAT shuffling the expression of different T-cell activation markers including the cytokines interleukin-2 and interferon-γ can be detected 24 h post irradiation with a significant expression increase compared to unirradiated controls. The X-ray triggered signaling events are similar to those observed in naive T-lymphocytes after TCR immune stimulation. This similarity is further highlighted by the fact that not only the key molecules, which are involved in the signaling cascades are identical, but also the dynamics of the events. This includes the delay times between stimulation and onset of Ca2+ oscillations and NFAT translocation as well as the highly conserved frequency of calcium oscillations. In conclusion the present data underscore the assumption that X-ray exposure of T-cells to clinically relevant doses induce an immunological stimulation of these cells via an activation of the same signaling cascade that is also elicited by TCR stimulation. This agonistic effect of X-ray exposure on T-cells might in the future foster therapy approaches which combine the effects of radiation with immunotherapy.