Ionizing radiation reduces the capacity of activated macrophages to induce T-cell proliferation, but does not trigger dendritic cell-mediated non-targeted effects.

PURPOSE: Previous investigations revealed influences of irradiation up to 2Gy on the cytokine secretion profile of inflammatory and peritoneal mouse macrophages (pMФ). This raised the question if those alterations impact on dendritic cells and consecutive T-cell responses. Further, the impact of irradiation directly on pMФ capacity to induce T-cell responses was analyzed. MATERIALS AND METHODS: pMФ were LPS-activated, irradiated and the expression of activation markers was assessed. Treated pMФ were co-incubated with T-cells to investigate proliferation. To verify modulating properties of pMФ supernatants isolated 24 h after irradiation, bone marrow-derived dendritic cells (BMDC) were co-incubated with supernatants and activation markers as well as the BMDC-induced proliferation of T-cells were measured. RESULTS: pMФ showed a highly significantly decreased major histocompatibility complexII (MHCII) expression within a dose range from 0.7-2Gy. Further, the proliferation rate of cluster of differentiation 4+ (CD4+) T-cells was decreased after co-incubation particularly with 2 Gy irradiated pMФ. The co-incubation of BMDC with supernatants of activated, irradiated pMФ significantly reduced the CD40 expression, but did not impact on the BMDC-derived induction of T-cell proliferation. CONCLUSIONS: Inflammatory macrophages being exposed to irradiation have the potential to modulate consecutive adaptive immune reactions. But supernatants of irradiated macrophages do not influence the dendritic cells (DC)-mediated induction of T cell proliferation.

Modulations in the Peripheral Immune System of Glioblastoma Patient Is Connected to Therapy and Tumor Progression-A Case Report from the IMMO-GLIO-01 Trial.

Immune responses are important for efficient tumor elimination, also in immune privileged organs such as the brain. Fostering antitumor immunity has therefore become an important challenge in cancer therapy. This cannot only be achieved by immunotherapies as already standard treatments such as radiotherapy and chemotherapy modify the immune system. Consequently, the understanding of how the tumor, the tumor microenvironment, and immune system are modulated by cancer therapy is required for prognosis, prediction, and therapy adaption. The prospective, explorative, and observational IMMO-GLIO-01 trial was initiated to examine the detailed immune status and its modulation of about 50 patients suffering from primary glioblastoma multiforme (GBM) or anaplastic astrocytoma during standard therapy. Prior to the study, a flow cytometry-based assay was established allowing the analysis of 34 immune cell subsets and their activation state. Here, we present the case of the first and longest accompanied patient, a 53-year-old woman suffering from GBM in the front left lobe. In context of tumor progression and therapy, we describe the modulation of the peripheral immune status over 17 months. Distinct immune modulations that were connected to therapy response or tumor progression were identified. Inter alia, a shift of CD4:CD8 ratio was observed that correlated with tumor progression. Twice we observed a unique composition of peripheral immune cells that correlated with tumor progression. Thus, following up these immune modulations in a closely-meshed manner is of high prognostic and predictive relevance for supporting personalized therapy and increasing therapy success. Clinical Trial registration: ClinicalTrials.gov, identifier NCT02022384 (registered retrospectively on 13th of December, 2013).

Modulation of the peripheral immune system after low-dose radon spa therapy: Detailed longitudinal immune monitoring of patients within the RAD-ON01 study.

The pain-relieving effects of low-dose radon therapies on patients suffering from chronic painful inflammatory diseases have been described for centuries. Even though it has been suggested that low doses of radiation may attenuate chronic inflammation, the underlying mechanisms remain elusive. Thus, the RAD-ON01 study was initiated to examine the effects of radon spa therapy and its low doses of alpha radiation on the human immune system. In addition to an evaluation of pain parameters, blood was drawn from 100 patients suffering from chronic painful degenerative musculoskeletal diseases before as well as 6, 12, 18, and 30 weeks after the start of therapy. We verified significant long-term pain reduction for the majority of patients which was accompanied by modulations of the peripheral immune cells. Detailed immune monitoring was performed using a multicolor flow cytometry-based whole blood assay. After therapy, the major immune cells were only marginally affected. Nevertheless, a small but long-lasting increase in T cells and monocytes was observed. Moreover, neutrophils, eosinophils and, in particular, dendritic cells were temporarily modulated after therapy. Regarding the immune cell subsets, cytotoxic T and NK cells, in particular, were altered. However, the most prominent effects were identified in a strong reduction of the activation marker CD69 on T, B, and NK cells. Simultaneously, the percentage of HLA-DR+ T cells was elevated after therapy. The RAD-ON01 study showed for the first time a modulation of the peripheral immune cells following standard radon spa therapy. These modulations are in line with attenuation of inflammation.

Interconnection between DNA damage, senescence, inflammation, and cancer.

In order to deal with endogenous and exogenous factors, including radiation or pathogens, cells evolved different strategies. This includes highly complex processes such as DNA damage response, senescence, cell death, and inflammatory reactions. Recent research indicates an interconnection between the mentioned cellular pathways whilst all of them seem to play a role in induction and progression, but also the prevention of cancerous diseases and therefore qualify for potential prevention and treatment strategies. On the basis of their pivotal functions in cancer biology in general, each of the cellular processes represents promising single therapeutic targets. Further, due to their strong interconnection, targeting all of them in a multimodal approach could be another promising strategy to treat cancer. We, therefore, review the mechanisms of DNA damage induction, detection and repair as well as the induction of cell death. Further, features of senescence and mechanism of inflammation induction and abrogation are outlined. A special focus is set on how senescence and inflammation are related to diseases and how targeting them could contribute to improvement of cancer therapies.

Cancer Cell Death-Inducing Radiotherapy: Impact on Local Tumour Control, Tumour Cell Proliferation and Induction of Systemic Anti-tumour Immunity.

Radiotherapy (RT) predominantly is aimed to induce DNA damage in tumour cells that results in reduction of their clonogenicity and finally in tumour cell death. Adaptation of RT with higher single doses has become necessary and led to a more detailed view on what kind of tumour cell death is induced and which immunological consequences result from it. RT is capable of rendering tumour cells immunogenic by modifying the tumour cell phenotype and the microenvironment. Danger signals are released as well as the senescence-associated secretory phenotype. This results in maturation of dendritic cells and priming of cytotoxic T cells as well as in activation of natural killer cells. However, RT on the other hand can also result in immune suppressive events including apoptosis induction and foster tumour cell proliferation. That's why RT is nowadays increasingly combined with selected immunotherapies.

Reduced secretion of the inflammatory cytokine IL-1ß by stimulated peritoneal macrophages of radiosensitive Balb/c mice after exposure to 0.5 or 0.7 Gy of ionizing radiation.

Since the beginning of the 20th century, low dose radiotherapy (LD-RT) has been practiced and established as therapy of inflammatory diseases. Several clinical studies already have proven the anti-inflammatory effect of low doses of ionizing irradiation (LDR). However, further research is inevitable to reveal the underlying immune-biological mechanisms. Focus has been set on the modulation of activated macrophages by LDR, since they participate in both, initiation and resolution of inflammation. Here we examined with an ex vivo peritoneal mouse macrophage model how LDR modulates the secretion of the inflammatory cytokines IL-1ß and TNF-α by activated macrophages and whether the basal radiosensitivity of the immune cells has influence on it. Peritoneal macrophages of Balb/c mice responded to exposure of 0.5 or 0.7 Gy of ionizing irradiation (X-ray) with significant decreased release of IL-1ß and slightly, but not significantly, reduced release of TNF-α. Macrophages of the less radiosensitive C57BL/6 mice did not show this anti-inflammatory reaction. This was observed in both wild type and human TNF-α transgenic animals with C57BL/6 background. We conclude that only the inflammatory phenotype of more radiosensitive macrophages is reduced by LDR and that ex vivo and in vivo models with primary cells should be applied to examine how the immune system is modulated by LDR.

Selected anti-tumor vaccines merit a place in multimodal tumor therapies.

Multimodal approaches are nowadays successfully applied in cancer therapy. Primary locally acting therapies such as radiotherapy (RT) and surgery are combined with systemic administration of chemotherapeutics. Nevertheless, the therapy of cancer is still a big challenge in medicine. The treatments often fail to induce long-lasting anti-tumor responses. Tumor recurrences and metastases result. Immunotherapies are therefore ideal adjuncts to standard tumor therapies since they aim to activate the patient's immune system against malignant cells even outside the primary treatment areas (abscopal effects). Especially cancer vaccines may have the potential both to train the immune system against cancer cells and to generate an immunological memory, resulting in long-lasting anti-tumor effects. However, despite promising results in phase I and II studies, most of the concepts finally failed. There are some critical aspects in development and application of cancer vaccines that may decide on their efficiency. The time point and frequency of medication, usage of an adequate immune adjuvant, the vaccine's immunogenic potential, and the tumor burden of the patient are crucial. Whole tumor cell vaccines have advantages compared to peptide-based ones since a variety of tumor antigens (TAs) are present. The master requirements of cell-based, therapeutic tumor vaccines are the complete inactivation of the tumor cells and the increase of their immunogenicity. Since the latter is highly connected with the cell death modality, the inactivation procedure of the tumor cell material may significantly influence the vaccine's efficiency. We therefore also introduce high hydrostatic pressure (HHP) as an innovative inactivation technology for tumor cell-based vaccines and outline that HHP efficiently inactivates tumor cells by enhancing their immunogenicity. Finally studies are presented proving that anti-tumor immune responses can be triggered by combining RT with selected immune therapies.

How does ionizing irradiation contribute to the induction of anti-tumor immunity?

Radiotherapy (RT) with ionizing irradiation is commonly used to locally attack tumors. It induces a stop of cancer cell proliferation and finally leads to tumor cell death. During the last years it has become more and more evident that besides a timely and locally restricted radiation-induced immune suppression, a specific immune activation against the tumor and its metastases is achievable by rendering the tumor cells visible for immune attack. The immune system is involved in tumor control and we here outline how RT induces anti-inflammation when applied in low doses and contributes in higher doses to the induction of anti-tumor immunity. We especially focus on how local irradiation induces abscopal effects. The latter are partly mediated by a systemic activation of the immune system against the individual tumor cells. Dendritic cells are the key players in the initiation and regulation of adaptive anti-tumor immune responses. They have to take up tumor antigens and consecutively present tumor peptides in the presence of appropriate co-stimulation. We review how combinations of RT with further immune stimulators such as AnnexinA5 and hyperthermia foster the dendritic cell-mediated induction of anti-tumor immune responses and present reasonable combination schemes of standard tumor therapies with immune therapies. It can be concluded that RT leads to targeted killing of the tumor cells and additionally induces non-targeted systemic immune effects. Multimodal tumor treatments should therefore tend to induce immunogenic tumor cell death forms within a tumor microenvironment that stimulates immune cells.

Old and new facts about hyperthermia-induced modulations of the immune system.

Hyperthermia (HT) is a potent sensitiser for radiotherapy (RT) and chemotherapy (CT) and has been proven to modulate directly or indirectly cells of the innate and adaptive immune system. We will focus in this article on how anti-tumour immunity can be induced by HT. In contrast to some in vitro assays, in vivo examinations showed that natural killer cells and phagocytes like granulocytes are directly activated against the tumour by HT. Since heat also activates dendritic cells (DCs), HT should be combined with further death stimuli (RT, CT or immune therapy) to allocate tumour antigen, derived from, for example, necrotic tumour cells, for uptake by DCs. We will outline that induction of immunogenic tumour cells and direct tumour cell killing by HT in combination with other therapies contributes to immune activation against the tumour. Studies will be presented showing that non-beneficial effects of HT on immune cells are mostly timely restricted. A special focus is set on immune activation mediated by extracellular present heat shock proteins (HSPs) carrying tumour antigens and further danger signals released by dying tumour cells. Local HT treatment in addition to further stress stimuli exerts abscopal effects and might be considered as in situ tumour vaccination. An increased natural killer (NK) cell activity, lymphocyte infiltration and HSP-mediated induction of immunogenic tumour cells have been observed in patients. Treatments with the addition of HT therefore can be considered as a personalised cancer treatment approach by specifically activating the immune system against the individual unique tumour.

Low dose ionising radiation leads to a NF-κB dependent decreased secretion of active IL-1ß by activated macrophages with a discontinuous dose-dependency.

PURPOSE: Therapy with low doses of ionising radiation (X-rays) exerts anti-inflammatory effects. Little is known about whether and how low doses of X-ray treatment modulate the inflammatory phenotype of macrophages, especially the secretion of Interleukin-1beta (IL-1ß). MATERIALS AND METHODS: Macrophages were differentiated from human THP-1 monocytes, activated with lipopolysaccharide (LPS), treated with distinct low doses of X-rays, and co-activated with monosodium urate crystals (MSU) to induce inflammasome activation. Secretion of IL-1ß was analysed by an enzyme-linked immunosorbent assay (ELISA) and Western blot. Furthermore, we analysed the intracellular amounts of the serine/threonine protein kinase B (named: Akt), mitogen-activated protein kinase p38 (p38), the v-rel reticuloendotheliosis viral oncogene homolog A (RelA), and pro- and cleaved IL-1ß. RESULTS: Low dose X-rays led to decreased secretion of active IL-1ß in a manner discontinuous with dose which was most pronounced after 0.5 or 0.7 Gy. Passive release of lactate dehydrogenase (LDH) was not influenced by X-rays. The decreased secretion of IL-1ß correlated with reduced translocation of RelA, being part of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) complex, into the nucleus. After 0.5 or 0.7 Gy of X-rays, the intracellular protein amounts of up (p38) and downstream molecules (Akt) of NF-κB were reduced in activated macrophages, as were the pro- and cleaved forms of IL-1ß. CONCLUSIONS: Distinct low doses of X-rays induce an anti-inflammatory phenotype of activated macrophages by lowering the amount of secreted IL-1ß in a NF-κB dependent manner.