Cancer Letters 356 (2015) 105–113

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Mini-review

Contribution of the immune system to bystander and non-targeted effects of ionizing radiation Franz Rödel a,⇑, Benjamin Frey b, Gabriele Multhoff c,d, Udo Gaipl b a

Department of Radiotherapy and Oncology, Goethe-University Frankfurt am Main, Frankfurt am Main, Germany Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany c Department of Radiation Oncology, Technical University of Munich, Munich, Germany d CCG ‘‘Innate Immunity in Tumor Biology’’, Helmholtz Zentrum München (HMGU), Munich, Germany b

a r t i c l e

i n f o

Article history: Received 9 July 2013 Received in revised form 13 August 2013 Accepted 11 September 2013

Keywords: Ionizing radiation Bystander effects Non-targeted effects Abscopal effect Immune system Heat shock protein 70

a b s t r a c t Considerable progress has recently been achieved in the understanding of molecular mechanisms involved in cellular radiation responses and radiation mediated microenvironmental communication. In line with that, it has become more and more obvious that X-irradiation causes distinct immunological effects ranging from anti-inflammatory activities if applied at low (50–100%) without induction of radiogenic acute or chronic side effects (outlined in more detail in [8]). On the contrary, LD-RT is still considered unfashionable in some countries due to reports of harmful late effects and increased mortality from leukaemia and anaemia published in the 1960s [12,13]. Pharmaceutical alternatives such as non-steroidal or steroidal drugs, however, also display numerous severe side effects and a considerable number of patients fail to respond to treatment [14]. Although a carcinogenic risk of low-dose irradiation is still a matter of controversy, improved radiation protection and recent progress in the development of predictive objectives for the response to LD-RT such as pre-treatment sonographic classification of calcifying tendonitis [15] may help to reconsider LD-RT as an effective treatment. In line with that, a recent patterns-of-care study reported 95% referral for radiation therapy in 4500 patients with osteoarthritis of the knee demonstrating an increased acceptance. Finally, Ott and colleagues recently reported that radiation therapy with lower single doses of 0.5 Gy might be as equally effective as single doses of 1.0 in the treatment of painful elbow or shoulder syndrome thus substantially decreasing the potential radiation risk [16,17]. By contrast, underlying radiobiological and immunological mechanisms are far from being fully explored. During the last two decades, however, the modulation of a multitude of inflammatory processes by LD-RT has been reported in vitro and in vivo [9]. These include modulation of mononuclear/polymorph nuclear leukocyte functions, apoptosis regulation, leukocyte/endothelial cell interaction, as well as surface marker and cytokine/chemokine expression (Fig. 1).

2.1. Non-targeted effects in leukocytes contribute to the antiinflammatory effects of low dose irradiation Whereas different lineages of lymphocytes (B and T cells) comprise cellular members of an antigen-specific effector response, polymorph nuclear cells (PMN: neutrophilic, eosinophilic and basophilic granulocytes) and peripheral blood mononuclear leukocytes (PBMC) are major components of the innate immune system representing the first line of host immune defence [18]. Due to their central role in the initiation and the resolution of an inflammatory process, mononuclear leukocytes (macrophages and dendritic cells) derived from peripheral blood precursors are considered as key players in the regulation of inflammation and immune responses [19]. Tissue resident macrophages, for example, support a local inflammatory process by a multitude of functions including phagocytosis, antigen presentation, secretion of cytokines, release of reactive oxygen intermediates (ROIs), and the expression of enzymes like inducible nitric oxide synthase (iNOS) [20]. iNOS processes the synthesis of nitric oxide (NO) that in turn increases vascular permeability and is involved in inflammatory pain [21]. In that context, low dose irradiation (61.0 Gy) decreases iNOS protein and NO production without affecting iNOS mRNA expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) and interferon-c (IFN-c) [22]. This may indicate a post-translational regulation of the enzyme that is linked to the analgesic properties of LD-RT. Furthermore, low dose X-irradiation significantly reduced oxidative burst capacity in murine RAW 264.7 macrophages after stimulation with tumour necrosis factor-a (TNF-a)/(IFN-c), Phorbol 12-myristate 13-acetate (PMA) or the yeast product Zymosan, whereas elevated doses had little effect [23]. This further highlights the therapeutic effect and modulation of an anti-inflammatory microenvironment by low dose irradiation.

Fig. 1. Current model on cellular compounds and factors involved in a local antiinflammatory activity of irradiation. Exposure of activated ECs to a dose of 0.3– 0.5 Gy resulted in a modulation of miRNA and XIAP expression and as a consequence increased NF-jB activity, increased TGF-b1 expression and a reduced peripheral blood mononuclear cell (PMBC)/endothelial cell (EC) adhesion. In leukocytes an induction of apoptosis, a reduced secretion of the chemokine CCL20, a hampered activity of the iNOS-pathway, a lowered oxidative burst, a decreased secretion of the pro-inflammatory cytokines IL-1b and TNF-a from activated macrophages as well as a lowered expression of MAPK (p38, Akt) may contribute to anti-inflammatory effects.

Further, a pivotal molecular mechanism in the regulation of an irradiation associated inflammatory, stress or bystander response includes the expression and secretion of regulatory peptides namely cytokines, chemokines and growth factors. While inflammation and immune activation promoting factors such as interleukin-1 (IL-1), TNF-a and chemotactic factors (e.g. IL-8 and CCL20) activate the immune system, anti-inflammatory factors such as the isoforms of transforming growth factor (TGF)-b1-3 or IL-10 limit immune responses and inflammatory cascades [24–26]. With regard to cytokine production, a hampered pro-inflammatory TNF-a secretion at doses of 0.5 Gy and 0.7 Gy from human THP-1 derived or RAW 264.7 macrophages stimulated by LPS was reported by Tsukimoto [22] and an additional reduced secretion of the pro-inflammatory cytokine IL-1-b was observed in RAW 264.7 macrophages which have been co-activated with mono sodium urate crystals (MSU) [21]. Mechanistically, the hampered cytokine production correlates with a diminished nuclear translocation of the immune relevant transcription factor nuclear factorjB (NF-jB) subunit RelA (p65) [21] in line with a decreased expression of NF-jB upstream (p38 mitogen activated protein kinase (MAPK)) and downstream factors like Protein Kinase B (Akt). Additionally, a dephosphorylation of both extracellular-signal-regulated kinases 1/2 (ERK1/2) and p38 MAPK was observed as early as 15 min after a 0.5 Gy X-ray exposure concomitant with a significantly increased expression of MAPK phosphatase-1 (MKP-1) [27]. Recently, Frischholz and colleagues reported that peritoneal macrophages derived from radiation sensitive Balb/c mice respond to a 0.5 or 0.7 Gy exposure with a diminished IL-1b and TNF-a release, whereas macrophages form less radiosensitive C57/BL6 mice did not [28]. This further highlights the complex regulation of low dose irradiation mediated cytokine expression and an involvement of genotype-dependent mechanisms that foster continuative investigations. Neutrophilic PMN infiltration has been implicated in the pathophysiology of acute and chronic inflammatory diseases, such as rheumatoid arthritis [29], in part by the secretion of chemokines to amplify and direct leukocyte infiltration [30]. Irradiation with doses between 0.5 and 1 Gy resulted in a significant reduction of CCL20 chemokine release from PMN in parallel to a significant

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reduction in PMN/EA.hy.926 endothelial cell (EC) adhesion. Moreover, as compared to CXCL8 and CCL18, CCL20 secretion is exclusively induced by a direct cellular contact between PMN and Ea.Hy.926 EC in a TNF-a dependent, but intercellular adhesion molecule 1 (ICAM-1)-independent manner [31]. Apoptosis, a physiological endogenous cellular suicide program induced by both endogenous and exogenous stimuli [32], significantly impacts on cellular homeostasis, immune regulation, and radiation response and is crucially involved in non-targeted effects. It should be stressed in that context that activated mononuclear cells reduce secretion of pro-inflammatory cytokines (e.g. TNF-a or IL-1) in the presence of apoptotic cells but respond by the production of anti-inflammatory cytokines like IL-10 indicating immunosuppressive properties of dying cells [33]. In this regard, a discontinuity (plateau or peak) in apoptosis induction in PBMC following irradiation with 0.3 Gy and 0.7 Gy was observed [34], that may well contribute to the above-mentioned suppressive characteristics of LD-RT. Moreover, a time-dependent loss of surface marker L-selectin by proteolytic shedding that is associated with their early apoptotic phenotype [35] augments the reduction in PBMC/EC adhesion and further contributes to the anti-inflammatory effect of low-dose irradiation. PBMC were X-irradiated with 0.1, 0.5 and 3 Gy and cell-free conditioned medium from these irradiated cells was transferred to unexposed lymphocytes (classical bystander experiments). Following 48 h of medium transfer a significant decrement in cellular viability, loss of mitochondrial membrane potential, and increased apoptosis compared to non-exposed lymphocytes became evident [36], inpointing a highly complex bystander regulation of apoptosis in this dose range. A non-linear appearance of apoptotic cell death displaying a relative maximum at 0.3 Gy and minimum at 0.5 Gy was further reported in PMN irradiated 2 h before stimulation with PMA [37]. Notably, this behaviour coincided with decreased protein levels of total cellular mitogen activated protein (MAP) kinases and Akt, shown to be involved in pro-survival pathways, proliferation, and transcription [38]. Finally, Rastogi et al. very recently reported that in vivo low dose radiation exposure is associated with a genotype-dependent macrophage activation, cytokine signalling and ROS/NO production. The group reported that macrophages from CBA/Ca mice exhibit an M1 (pro-inflammatory) phenotype as compared to M2 (anti-inflammatory) macrophages derived from C57BL/6 mice [39]. Moreover, the ability of macrophages to interact with apoptotic cells varies considerable according to the macrophage phenotype. M2 macrophages induce anti-inflammatory markers such as arginase and TGF-b after engulfment of apoptotic cells. In contrast, M1 macrophages express pro-inflammatory markers like IL-6, TNFa, superoxide and NO. These results further indicate a complex cross talk between macrophages and apoptotic cells and demonstrate that phagocytic clearance of apoptotic cells may contribute to microenvironmental responses that differ in sensitive CBA/Ca mice with M1 macrophage activation, but not in resistant C57BL/ 6 mice with M2 macrophage activation.

2.2. Non-targeted effects in endothelial cells contribute to immune modulatory properties of LD-RT Beside the different lineages of leukocytes as reported before, EC are crucially involved in the regulation of inflammatory processes both by a local recruitment of immune cells from the peripheral blood (adhesion) and their capacity to secrete a multitude of cytokines/chemokines [40]. This prompted us to analyse putative non-targeted effects on ECs in the immune modulatory properties of LD-RT.

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Applying adhesion assays using human Ea.Hy.926 or murine (mlEND1) EC low dose X-irradiation prior to stimulation by TNFa resulted in a dose-dependent reduction of leukocyte adhesion to 43–50% of the control level at 4 h and 24 h but elevated values at 12 h after irradiation most pronounced after irradiation with a dose of 0.5 Gy [41–43]. This characteristic coincides with a biphasic expression of the well-known anti-inflammatory and bystander cytokine TGF-b1 from EC. In line with that, neutralizing TGF-b1 by antibody treatment restored adhesion of PBMC to irradiated EC [42], highlighting its key role in the modulation of the adhesion process following low dose X-ray exposure. A hampered adhesion and consequently reduced immune cell infiltration [44] is further supported by a lowered expression of the surface adhesion molecule E-selectin on stimulated EC with a local minimum following in the dose range of 0.3–0.5 Gy [42,43]. There is substantial evidence for a mechanistic link between transcription factors including tumour suppressor protein 53 (TP53), activating protein 1 (AP-1), and NF-jB in cellular radiation response, immune regulation, as well as in carcinogenesis [45–47]. Thus, it is likely that NF-jB also participates in non-targeted or bystander effects and displays a central linker between inflammation, immune regulation and irradiation effects. In that context, activation of NF-jB in a discontinuous manner with peak activities at 8 h and 36 h after irradiation was reported in 244B lymphoblastoid and B16 melanoma cells [48]. In accordance with these findings, a non-linear dose and time kinetics of NF-jB DNA binding and transcriptional activity was confirmed in stimulated Ea.Hy.926 EC peaking at 4 h and 24–30 h after irradiation, respectively [49]. Moreover, based on these initial data, factors involved in the regulation of NF-jB activation were explored in stimulated EA.Hy926 EC. Among these regulatory proteins, X chromosome-linked inhibitor of apoptosis protein (XIAP) that enhances NF-jB subunit RelA/ p65 nuclear translocation and promotes the degradation of NF-jB inhibitory protein IjB [50,51] was investigated in more detail. A discontinuous profile of XIAP protein expression was observed with a relative maximum following a 0.5 Gy and 3.0 Gy exposure. Furthermore, RNA-interference (siRNA)-derived attenuation of XIAP resulted in hampered NF-jB transcriptional activity, reduced secretion of TGF-b1 and abolishment of a diminished adhesion at 0.5 Gy indicating a regulatory molecular and functional interrelationship between these factors [52]. Apart from NF-jB activation, ionizing radiation activates the expression of immediate early genes encoding for c-Fos and c-Jun [47] that collectively form the homo- or heterodimeric AP-1 transcription factor complex [53]. By applying electrophoretic mobility shift assays (EMSA) and transcriptional activity assays, a biphasic induction of AP-1 with local maxima at 0.3 Gy was detected in EA.Hy926 EC that may further contribute to the immunomodulatory properties of LD-RT [54]. 2.3. Novel molecular aspects of the regulation of non-targeted effects in EC Although considerable progress has been achieved in the understanding of non-targeted effects being prominent after a low-dose exposure, the exact molecular mechanisms are presently not resolved. They may, however, originate from an overlap of several pathways that are initiated at various thresholds, display different kinetics, and operate in a staggered manner. These pathways may include a differential regulation of an ATM-dependent G2-phase cell cycle arrest and apoptosis induction, as proposed for the phenomenon of low-dose hyper-radiosensitivity (HRS) and induced radioresistance (IRR) [55], epigenetic alterations like DNA methylation, histone modification, and chromatin remodelling (reviewed in more detail in [56]) or altered protein expression and 26S proteasome activity [57].

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Applying in-gel trypsin digestion, Matrix-assisted Laser Desorption/Ionization (MALDI)–time of flight (TOF) tandem mass spectrometry, and peptide mass fingerprint analyses 15 differentially expressed proteins were identified following irradiation of Ea.Hy.926 EC, with 10 proteins up- and 5 down-regulated. Pathways influenced by the factors included the RhoA motility pathway, fatty acid metabolism and cellular stress response [58]. More recently, evidences for an involvement of various microRNAs in the regulation of inflammatory processes and cellular response towards low dose irradiation have been reported. MicroRNAs (miRNAs) are a class of non-coding RNA molecules of 21–23 base pairs that are emerging as key players in regulating gene expression at a post-transcriptional level by degradation of mRNA or translational attenuation [59]. Recent data further indicate that approximately 60% of all human coding genes are regulated by miRNAs [60] and that deregulation of miRNAs is connected to a broad range of malignant and inflammatory diseases such as rheumatoid arthritis [59]. Thus, one may speculate that a differential expression of miRNAs may contribute to the modulatory properties of EC. Indeed, alterations in endothelial miRNA expression patterns in response to ionizing radiation as well as an association between miRNA deregulation, apoptosis and cell cycle checkpoint activation in irradiated cells have recently been reported [61–63]. For instance, Barjaktarovic and colleagues reported miR-21 and miR-146b to be significantly deregulated in primary human coronary artery EC following a single 0.2 Gy irradiation in line with a negative correlation between miR-21 levels and predicted target proteins, desmoglein 1, phosphoglucomutase and target of Myb protein [64]. In summary these novel findings may well contribute to the regulation of local non-targeted/bystander responses and the modulation of local inflammatory processes by low dose irradiation.

3. Abscopal anti-tumour effects are immune mediated While chronic inflammation contributes to tumorigenesis [65] an acute one fosters anti-tumour immune responses [66]. The latter act systemically and can be induced by local irradiation of the tumour (Fig. 2). The appearance of systemic effects in non-irradiated areas after treatment with localized radiation therapy (RT) is called abscopal effect that occurs at a distance from the irradiated volume, but within the same organism [67]. But how can tumours inside and outside the irradiation field be recognised by immune cells when they have been formerly ignored? The current concept is based on the induction of immunogenic tumour cell death forms by standard anti-cancer treatments with distinct chemotherapeutic agents and/or RT [68]. Of note is that in the case of local RT the immune mediated abscopal effects are most pronounced after combined immune activation to stimulate dendritic cells (DCs) (summarised in [69,70]). DCs and macrophages, which engulf, process and present dying tumour cell material to adaptive immune cells trigger adaptive immune responses [71]. DCs are the most potent population of antigen presenting cells (APCs), act as messengers between the innate and adaptive immunity, and play a central role in tolerance induction and immunity. Tumour cell constituents are taken up and tumour-associated antigens are processed by DCs. In consequence, DCs mature and migrate into the draining lymph nodes. There, DCs present the tumour peptides via MHC complexes and deliver co-stimulatory signals to naive CD4 positive and CD8 positive T lymphocytes. The latter differentiate into cytotoxic T lymphocytes (CTLs) that specifically kill tumour cells. Demaria and co-workers demonstrated in the ectopic syngeneic mammary 67NR carcinoma mouse model that the growth of tumour masses outside the irradiation field was impaired after RT with a clinical relevant single dose of 2 Gy, but only in combination

Fig. 2. Beside local activities, out of (radiation) field distant bystander (abscopal) effects at higher doses do impact on non-irradiated tumour masses and originate at least in part from enhanced immune functions such as dendritic cell (DC) and cytotoxic T cell (CTL) activation. Mostly, combination(s) of radiation with further immune stimulation (immune therapy, IT) or distinct fractionation schemes such as hypofractionation with 6 Gy may lead to abscopal anti-tumour immune reactions.

with the application of the DC growth factor Fms-related tyrosine kinase 3 (Flt3)-Ligand as immune therapy (IT). The abscopal effects were tumour specific and were not observed in mice lacking functional T cells, suggesting that immune cells are crucial in eliciting distant bystander effects [72]. Similar results were observed when a higher single radiation dose of 6 Gy was used (hypofractionated RT). Again, the abscopal anti-tumour effects of RT occurred not until an additional immune activation was transmitted (Fig. 2). Moreover, the direct injection of DCs into an irradiated MCA-102 fibrosarcoma tumour resulted in the induction of anti-tumour immunity against a tumour established at a distant site [73] and this abscopal effect could even be enhanced by pre-treatment with low dose cyclophosphamide [74]. Distinct radiochemoimmunotherapy protocols are apparently useful for the induction of systemic anti-tumour immunity. Preclinical experiments in TSA mouse breast carcinoma and a MCA38 mouse colon carcinoma model revealed once more that only combination of RT with further immune activation, in this case by blocking immune inhibitory signals on T cells with an antibody against CTLA-4, results in abscopal and T cell mediated anti-tumour effects. The growth delay of the tumour outside the irradiated field correlated with the frequency of CD8+ T cells and occurred only after fractionated RT (3  8 Gy or 5  6 Gy), but not after single irradiation with a high dose of 20 Gy, in combination with IT. The general anti-tumour effect of RT was demonstrated to be almost completely abolished in tumour-bearing mice receiving antibodies that deplete CD8+ T cells. This indicates that tumour-specific cytotoxic T-lymphocytes (CTLs) play a crucial role in the tumour growth retardation induced by RT [75]. Local RT with either single (15 Gy) or fractionated (5  3 Gy) doses increases the generation of tumour antigen-specific effector cells as well as their trafficking into the tumour [76]. IFN-gamma in the tumour microenvironment after RT plays a central role in this connection [77,78]. Lee and colleagues demonstrated that ablative RT with a single dose of 15–25 Gy generates CD8+ T cell responses that are strong enough to induce reduction of the primary tumour. However, the eradication of metastasis being outside the irradiation field was most effective when combining RT with ad-LIGHT virus immune therapy that generates more CTLs [79,80]. Besides CTLs, CD4+ T cells and NK1.1 are involved in the immune mediated abscopal effects of RT in combination with immune activation, as shown for the chemokine macrophage inflammatory protein-1 alpha variant [1]. Combination of fractionated RT with a single dose of 2.5 Gy, but also with a high single dose of 20 Gy, with local low dose application of

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the cytokine IL-2 also resulted, besides increased local tumour control, in regression of distant, non-irradiated tumours [81]. The preclinical studies presented here indicate that there is a still an unsolved challenge to identify which single radiation dose and fractionation scheme is the most beneficial in induction of systemic anti-tumour immunity alone or especially when combined with certain immune therapies. Our own work give hints that normo- and hypofractionated irradiation of human colorectal tumour cells, but not a single high dose irradiation, creates tumour cell supernatants that activate DCs (Kulzer et al., unpublished data). Focus was set in this in vitro study on human monocyte-derived DCs and on an early time point (1 day) after the last irradiation. However, when RT is combined with further immune activation, such as hyperthermia, single low (2 Gy or 5 Gy) and high doses (10 Gy) modify the phenotype of colorectal tumour cells in a way that maturation markers and homing receptors are increased on DCs after contact with the therapy-tumour modified cells [82]. The latter release danger signals such as adenosine triphosphate (ATP), high mobility group box 1 protein (HMGB1) and/or heat-shock protein70 (Hsp70; see below) that play a pivotal role in triggering anti-tumour immune responses [83–85]. In summary, preclinical in vitro and in vivo models have convincingly demonstrated that local RT has the potential to induce abscopal anti-tumour immune effects (Fig. 2), but these clearly dependend of further immune stimulation and the composition of the tumour microenvironment (outlined in [86]).

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regression [95]. Thus, future randomized clinical trials should focus on distant bystander effects of RT in addition to other endpoints. Furthermore, strong efforts should be made to identify the optimal combination of RT (including different fractionation concepts), chemotherapy, and immune therapy as well as their chronology for the induction of specific and systemic immune-mediated anti-tumour responses in preclinical animal model systems. 4. Bystander and non-targeted effects on the immune system: stress response and heat shock proteins Apart from surgery and chemotherapy (CT), RT is one of the three hallmarks in cancer therapy. In recent years, innovative developments in instrumentations for a more precise delivery of irradiation, the utilization of protons and heavy ions and the emphasis on hypofractionated irradiation schemes resulted in an improved clinical outcome after RT. However, efficacy of radiotherapy is limited in locally advanced tumours and differs considerable between individual patients due to the heterogeneity of tumours and the tumour micromilieu (outlined in [96]). In order to provide the basis for the development of innovative combined therapeutic concepts consisting of radiochemotherapy (RCT) and immune therapy in patients with locally advanced cancer, it is essential to gain further insight into non-targeted effects on the tumour and its surrounding tissue. Thus, we aimed to review in more detail the role of the cellular stress response and heat shock proteins.

3.1. Clinical observation of abscopal effects An abscopal regression of hepatocellular carcinoma after RT with a total dose of 36 Gy for bone metastasis was observed and correlated with increased serum levels of the inflammatory cytokine TNF-a [87]. One cannot dissemble that at present mostly case reports about abscopal anti-tumour immune effects in the clinics have been published. In a patient with hepatocellular carcinoma, Okuma and colleagues observed a spontaneous shrinking of a lung metastasis which was located outside the irradiation field after radiotherapy of the mediastinum with 2.25 Gy per fraction [88]. In metastatic renal cell carcinoma, induction of anti-tumour responses against untreated metastases after extracranial stereotactic RT was observed in four cases of a total of 28 treated patients [89]. This highlights that immune mediated abscopal effects of RT are not a general phenomenon, but strongly depend on the tumour microenvironment that itself is dependent on multiple factors such as therapy-induced cell death forms and immune cell modulating chemokines and cytokines [90]. An early report of the 1980s already demonstrated in two patients with clinical Stage III non-Hodgkin’s lymphoma a reduction in the size of the abdominal lymph nodes following irradiation to the mantle [91]. Further, abscopal effects on metastatic lymph nodes after pre-operative RT in breast cancer was observed in at least 15 out of 42 cases [92]. An abscopal effect after six courses of chemotherapy plus 40 Gy involved field irradiation was even observed in natural killer cell lymphoma accompanied with massive CTLs infiltration in the relapsed lesions [93]. A case of an abscopal effect in malignant melanoma was described by Kingsley already in 1975 [94]. Now, first promising studies are coming up demonstrating immune mediated abscopal anti-tumour effects after combination of RT with immune therapy. Such multimodal treatments resulted in the most effective abscopal anti-tumour immune responses in most preclinical models (see above). Just recently, Postow and colleagues reported on abscopal effects in a patient with melanoma treated with Ipilimumab, an anti-CTLA-4 antibody, and RT. An increase of HLA-DR expressing monocytes and a decrease of immune suppressive myeloid-derived suppressor were observed in the peripheral blood of the patient that preceded radiographic disease

4.1. Ionizing radiation and stress-response, the role of heat shock proteins Heat shock proteins (HSP) are evolutionary highly conserved proteins that are expressed in a wide range of species from bacteria to men. According to their molecular weights that are ranging from approximately 20 to 100 kDa they are grouped into different families. Environmental stress such as ionizing irradiation, heat, hypoxia, heavy metals, oxidative stress as well as viral and bacterial infections induce an increased synthesis of Hsp [97,98] whereas synthesis of other proteins in general is down-regulated. The rapid induction of HSP in response to stress is based on a variety of genetic and biochemical processes referred to as the heat shock response (HSR; [99]) mainly regulated at the transcriptional level by heat shock factors (HSF). Among them, HSF-1 is considered as being the key factor of stress-inducible HSP [100,101]. A link between HSR and cancer development has been emerging since more than 10 years [102] as Hsps are over-expressed in a wide spectrum of human malignancies contributing to tumour growth, differentiation, invasiveness, and metastasis and being associated with poor prognosis (Fig. 3) [103–105]. Moreover, HSP overexpression in tumour cells is shown to play a pivotal role in tumorigenesis by inhibiting apoptosis and senescence. In breast cancer, transformation-induced activation of HSF-1 results in an up-regulated expression of Hsp27 and Hsp70 which in turn results in protection against apoptosis [106]. HSF-1 also triggers expression of Hsp90, an essential factor in tumour growth due to its ability to chaperone a variety of oncogenic signalling proteins including Her-2/neu (c-ErbB-2) and c-Src [106,107]. Since HSP overexpression also protects from drug-related apoptosis [108], these mechanisms highlight the role of HSP in tumour progression and therapy resistance. HSP protect cells against the lethal damage induced by exogenous and endogenous stress by supporting protein degradation, folding, transport of newly produced polypeptides and aberrantly folded proteins [109]. More recently, HSP were also found to be localized in the extracellular space of tumour cells, although the mechanism of transport has not been completely elucidated. Depending on their intra- and extracellular localization HSP

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mediate either protective or immune stimulatory functions, respectively. HSP with molecular weights of 70 and 90 kDa are found on the cell surface of tumour cell lines as determined by selective cell surface protein profiling [110]. In line with these results a screening of human tumour biopsies and normal tissues revealed a membrane positive phenotype selectively on tumour but not on non-cancerous cells [111]. The major stress-inducible Hsp70 is located on the plasma membrane in 50–70% of colon, lung, pancreas, mammary, head and neck, lung and urogenital carcinomas as determined by using a highly specific cmHsp70.1 monoclonal antibody [112–115]. After orthotopic injection of human tumour cells into immune deficient animals the cell surface density of Hsp70 was increased on metastases as compared to the primary tumours [116,117] and patients suffering from Hsp70 membrane positive tumours had a lower overall survival compared to those with Hsp70 negative tumours. These findings might reflect a more aggressive tumour phenotype in membrane Hsp70 positive malignancies [118]. Apart from solid tumours also bone marrow samples of patients suffering from acute (AML) and chronic (CML) myeloid leukaemia are frequently membrane Hsp70-positive [119]. A broad lipid profiling of isogenic tumour cells with differential Hsp70 membrane phenotype revealed that Hsp70 is anchored in the plasma membrane by the glycosphingolipid Gb3 that is expressed in a tumour-selective manner [120]. Thus, one can speculate that an association of Hsp70 with Gb3 already occurs in the cytosol and by fusion of Hsp70 containing intracellular lipid vesicles with the plasma membrane Hsp70 is transported to the cell surface. 4.2. Role of HSP in the induction of cancer immunity after irradiation Ionizing irradiation is a well-known stress factor that induces the synthesis, membrane transport and release of HSP. Tumour, in contrast to normal cells, exhibit higher basal Hsp70 and Hsp90 levels and following stress such as RT these levels further increase in the cytosol, the membrane and in the extracellular milieu of tumour cells. The pioneering work of the group of Srivastava et al. has demonstrated that Hsp70 and Hsp90 (gp96)-chaperoned peptides can be recognized by CD8-positive cytotoxic T lymphocytes after cross-presentation on major histocompatibility complex (MHC) class I molecules and thus generate a T cell mediated anti-cancer immune response [121]. Our group could show that even in the absence of immunogenic peptides, Hsp70 or a peptide derived thereof in combination with pro-inflammatory cytokines, such as IL-2 and IL-15 has the capacity to stimulate the cytolytic activity of

NK cells against highly aggressive membrane Hsp70-positive tumour cells [122,123]. Tumour cells that had been irradiated before at sub-lethal doses exhibit higher Hsp70 membrane densities and therefore, provide better targets for the cytolytic attack mediated by activated NK cells (Fig. 3). The mechanism of Hsp70 mediated tumour cell killing was identified as a perforin-independent, granzyme B-mediated cell death [124]. Granzyme B derived from activated NK cells specifically binds to membrane Hsp70 on tumour cells and following Hsp70-mediated endocytosis apoptosis is induced [125,126]. Incubation of NK cells with either full length Hsp70 protein or a 14mer-peptide derived from the C-terminus of Hsp70 is accompanied by an up-regulation of activating receptors on NK cells (CD94/ NKG2C, NKG2D, NKp30, NKp44, NKp46) [124,126] and Hsp70 membrane-positive tumours are eliminated by NK cells that had been pre-stimulated with low dose IL-2 plus Hsp70 peptide, in vitro [127]. Apart from that the cytolytic activity, the migratory capacity of activated NK cells was increased towards Hsp70-postive tumour cells and supernatants derived thereof [128]. The adoptive transfer of Hsp70 peptide-stimulated NK cells in tumour-bearing mice induces tumour cell regression and an increased overall survival in mice [116,129,130]. Regarding these results a clinical phase I trial has been initiated using ex vivo Hsp70 peptide TKD plus IL-2 activated autologous NK cells in patients with colorectal and non-small lung cell carcinomas (NSCLC). This trial showed excellent tolerability, feasibility and safety of TKD/IL-2 activated autologous NK cells [131]. Moreover, it is well established that IL-2 activated NK cells are able to induce regression of lung and liver tumours [132–134]. At present a phase II proof-of-concept trial is ongoing with the aim to show efficacy of ex vivo TKD/IL-2-stimulated, adoptively transferred, autologous NK cells in Non-small-cell lung carcinoma patients following combined radiochemotherapy. Further, an involvement of Hsp70 and Hsp90 in the recognition of Pathogen-Associated Molecular Pattern molecules (PAMP) by binding to Toll-like receptor 4 (TLR-4) has been demonstrated [135,136]. Since extracellular residing Hsp70 acts as a danger signal for the immune system [137], Hsp70 has been termed in line with HMGB1 as an ‘alarmin’. Alarmins and PAMPs [138] both belong to the group of Danger-Associated Molecular Pattern molecules (DAMP; [139]). Importantly, peptide-free Hsp70, added exogenously, stimulates the production of pro-inflammatory cytokines TNF-a, IL-1b and IL-6 by antigen presenting cells [140–142]. In bronchial epithelial cells extracellular Hsp70 has been found to induce the production of IL-8 [143,144]. From these observations one can conclude that beside a more specific cellular response,

Fig. 3. Tumour specific membrane localisation of Hsp70 is associated with a more aggressive and metastatic tumour phenotype, and contributes to a therapy resistance by modulating apoptotic cell death. Moreover, extracellular and membrane exposed Hsp70 is involved in the induction of an anti-tumour immunity, in particular NK cell activation, and Hsp70 derived immunotherapy if combined with radio(chemo)therapy may increase therapeutic effectiveness and tumour eradication.

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Hsp70 modulates immune responses and, inflammation in a nonspecific manner via the induction of the release of cytokines and chemokines [145,146]. 5. Conclusions Ionizing radiation induces a variety of biological effects in irradiated cells and induces a cellular communication with non-irradiated local neighbours or distant cells that is referred to as bystander response and abscopal effects. Although considerable progress has been achieved during the last decades in the understanding of underlying mechanisms being prominent at a low and high dose radiation exposure [70], a multitude of unresolved questions exists that foster further investigations. For instance, do local and distant effects and the activation of an anti-tumour immunity by radiation share more common mechanisms of innate and adaptive immune responses or do they differ considerably? As inflammatory diseases and tumour immune biology are characterized by complex (patho)physiological relationships, intensive basic and clinical research efforts as well as the development of suitable preclinical models are seriously needed to recognize additional contributing factors and mechanisms and to define chronologies of immune mediated processes. This, however, may not only improve future radiation therapy of non-malignant diseases but may also impact on radiation protection and multimodal combined radiation and immunotherapy of cancer. Conflicts of interest The authors state that there is no conflict of interest. Acknowledgements This work was supported by the European Commission under contract FP7-249689 (European Network of Excellence, DoReMi), the German Federal Ministry of Education and Research (GREWIS, 02NUK017G/F and m4 cluster of excellence: 16EX1021E/J/R) and the German Research Foundation (DFG, SFB-824 and Graduate schools 1657 and 1660). References [1] K. Shiraishi, Y. Ishiwata, K. Nakagawa, S. Yokochi, C. Taruki, T. Akuta, K. Ohtomo, K. Matsushima, T. Tamatani, S. Kanegasaki, Enhancement of antitumor radiation efficacy and consistent induction of the abscopal effect in mice by ECI301, an active variant of macrophage inflammatory protein1alpha, Clin. Cancer Res. 14 (2008) 1159–1166. [2] T.K. Hei, H. Zhou, Y. Chai, B. Ponnaiya, V.N. Ivanov, Radiation induced nontargeted response: mechanism and potential clinical implications, Curr. Mol. Pharmacol. 4 (2011) 96–105. [3] C. Mothersill, C. Seymour, Radiation-induced non-targeted effects of low doses-what, why and how?, Health Phys 100 (2011) 302. [4] M.H. Barcellos-Hoff, How tissues respond to damage at the cellular level: orchestration by transforming growth factor-{beta} (TGF-{beta}), BJR Suppl. 27 (2005) 123–127. [5] O.V. Belyakov, S.A. Mitchell, D. Parikh, G. Randers-Pehrson, S.A. Marino, S.A. Amundson, C.R. Geard, D.J. Brenner, Biological effects in unirradiated human tissue induced by radiation damage up to 1 mm away, Proc. Natl. Acad. Sci. USA 102 (2005) 14203–14208. [6] A. Bertucci, R.D. Pocock, G. Randers-Pehrson, D.J. Brenner, Microbeam irradiation of the C. elegans nematode, J. Radiat. Res. 50 (Suppl. A) (2009) A49–A54. [7] J. Williams, Y. Chen, P. Rubin, J. Finkelstein, P. Okunieff, The biological basis of a comprehensive grading system for the adverse effects of cancer treatment, Semin. Radiat. Oncol. 13 (2003) 182–188. [8] M.H. Seegenschmiedt, H.B. Makoski, K.R. Trott, L.W.E. Brady, Radiotherapy for Non-Malignant Disorders, Springer Verlag, Berlin, Heidelberg, 2008. [9] F. Rodel, B. Frey, U. Gaipl, L. Keilholz, C. Fournier, K. Manda, H. Schollnberger, G. Hildebrandt, C. Rodel, Modulation of inflammatory immune reactions by low-dose ionizing radiation: molecular mechanisms and clinical application, Curr. Med. Chem. 19 (2012) 1741–1750. [10] Solokoff, Röntgenstrahlen gegen Gelenkrheumatismus, Wien. Med. Wochenschr. (1898) 570.

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